TRC8, a gene related to the hedgehog receptor, patched

ABSTRACT

The present invention provides the sequence for a novel gene called TRC8 which is located on chromosome 8. Various types of alterations in the gene have been shown to be associated with renal and thyroid tumors. One such alteration involves a 3;8 translocation which interrupts TRC8 and results in a fusion with the 3p14 gene, FHIT. Another alteration includes a mutation in the 5′ untranslated region of TRC8. Thus, the invention further provides sequences corresponding to the gene fusions created during the translocation, as well as the sequence of the gene containing the mutation in the 5′ region. The invention further provides methods for detecting alterations in TRC8 which have potential utility in the diagnosis of tumors.

RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/077,723, filed Mar. 12, 1998, this application beingincorporated herein by reference.

STATEMENT REGARDING GOVERNMENT RIGHTS

[0002] This invention was made with support from Grant Number CA 581875PQ awarded by the National Institutes of Health. Therefore, thegovernment has certain rights in this invention.

FIELD OF INVENTION

[0003] The present invention relates to alterations in a novel genewhich are associated with certain renal and thyroid tumors. As such, thepresent invention is directed to the field of molecular genetics oftumor formation. One such alteration involves a chromosomaltranslocation between chromosomes 3 and 8 (typically referred to simplyas t(3;8)). The 3;8 translocation results in the fusion of the novelgene TRC8 (Translocation in Renal Cancer from Chromosome 8) with a knowngene named FHIT (Fragile Histidine Triad). A mutation in the 5′untranslated region has also been associated with certain renal cellcarcinomas.

BACKGROUND OF THE INVENTION

[0004] The 3;8 chromosomal translocation, t(3;8)(p14.2;q24.1), wasdescribed in a family with classical features of hereditary renal cellcarcinoma (RCC), i.e., autosomal dominant inheritance, early onset andbilateral disease (see A. J. Cohen, et al., N. Engl. J Med. 301, 592-595(1979)). The translocation and RCC segregated concordantly and afollow-up analysis reported the occurrence of thyroid cancer in twotranslocation carriers with kidney cancer (F. P. Li, et al., Ann.Intern. Med 118, 106-111 (1993)). Frequent 3p loss of heterozygosity(LOH) in sporadic clear-cell RCC led to the initial assumption that acritical tumor suppressor gene would be located at 3p14. Identificationof the von Hippel-Lindau (VHL) gene at 3p25, frequently mutated in RCCs,provided an alternative explanation for at least some observed 3p LOHand Van den Berg et al. subsequently reported that region p21 may be aprimary target for 3p LOH. (A. van den Berg and C. H. Buys, Genes.Chromosomes. Cancer 19, 59-76 (1997)).

[0005] Within 3p14, Ohta et al. identified a putative tumor suppressorgene (TSG), FHIT, which was interrupted in its 5′ untranslated region bythe 3;8 translocation (M. Ohta, et al., Cell 84, 587-597 (1996)). Thehuman gene, like its yeast homologue, encodes di-adenosine (5 ′, 5″- p¹,P³-triphosphate) hydrolase activity. (L. D. Barnes, et al., Biochemistry35, 11529-11535 (1996)). Several reports have described FHIT alterationsin diverse carcinomas using nested reverse transcriptase-PCR (RT-PCR)(M. Ohta, et al., Cell 84, 587-597 (1996); G. Sozzi, et al., Cell 85,17-26 (1996); L. Virgilio, et al., Proc. Natl. Acad. Sci. U.S.A. 93,9770-9775 (1996); M. Negrini, et al., Cancer Res. 56, 3173 (1996); G.Sozzi, et al., Cancer Res. 56, 2472-2474 (1996)). Other results,however, have been contradictory.

[0006] In fact, several lines of evidence make FHIT an unlikely, or atleast suspect, causative gene in the hereditary t(3;8) family. Forexample, the possibility that FHIT functions as a tumor suppressor is atodds with its activity as a di-adenosine hydrolase, an unprecedentedtumor suppressor function (Barnes, L. D., et al., Biochemistry 35,11529-11535 (1996)). The lack of substantial mutations in tumorscombined with the fact that most FHIT abnormalities occur in thepresence of wild-type transcripts and result from low-abundance splicingalterations, similar to those seen for TSG101, further argues againstFHIT acting as a tumor suppressor (S. Thiagalingam, et al., Cancer Res.56, 2936-2939 (1996); K. M. Fong, et al., Cancer Res. 57, 2256-2267(1997); S. A. Gayther, et al., Oncogene 15, 2119-2126 (1997); F. Boldoget al., Hum. Mol. Genet. 6, 193-203 (1997); I. Panagopoulos, et al.,Genes. Chromosomes. Cancer 19, 215-219 (1997); and A. van den Berg, etal., Genes. Chromosomes. Cancer 19, 220-227 (1997); A. Latil, et al.,Oncogene 16, 1863 (1998)).

[0007] Moreover, there is little support for the involvement of FHIT inrenal cancers (See, A. van den Berg, et al., Genes Chromosomes Cancer19, 220-227 (1997); P. Bugert, et al., Genes Chromosomes Cancer 20, 9-15(1997)). Similarly, the reintroduction of FHIT into tumorigenic celllines was inconsistent in suppressing tumors, including the fact that ahydrolase “dead” mutant appeared active (Z. Siprashvili, et al., Proc.Natl. Acad. Sci. USA 94, 13771-13776 (1997)). Otterson et al. (J. Natl.Cancer Inst. 90, 426-432 (1998)) introduced FHIT into six carcinoma celllines and observed no effects on proliferation, morphology, cell-cyclekinetics, or tumorigenesis.

[0008] In earlier work, the present inventors also identified a seriesof 3p14 deletions, many not involving FHIT exons, which overlapped FRA3Bin various carcinoma cell lines (F. Boldog, et al., Hum. Mol. Genet. 6,193-203 (1997)). However, spontaneous deletions also were observed innontumor backgrounds. Thus, the close association of FHIT exon 5 withFRA3B suggested that its loss might be primarily related to genomicinstability, in contrast to negative selection during tumor development.Although another 3p14 gene might exist, sequence data totaling 160 kbfrom FRA3B (F. Boldog, et al., Hum. Mol. Genet. 6, 193-203 (1997)) (plusGenBank updates AF023460 and AF023461), together with 135 kb ofnonoverlapping sequence from Inoue et al. (Proc. Natl. Acad. Sci. U.S.A.94, 14584-14589 (1997)), failed to show any additional definitive genes.

[0009] It was also noted that FHIT, in one parotid adenoma, underwentfusion with the high mobility group protein gene (HMGIC), the causativegene in a variety of benign tumors (J. M. Geurts, et al., Cancer Res.57, 13-17 (1997)). That HMGIC was involved in translocations with otherunrelated genes indicated that FHIT could be a bystander in theFHIT/HMGIC fusion.

[0010] Given this evidence arguing against FHIT as the causative gene inthe hereditary t(3;8) family, there remained a need to identify the geneor genes involved in the 3;8 translocation that results in the formationof tumors, especially renal and thyroid cancers. Given thecorrespondence between the 3;8 translocation and certain tumors,identification of the gene involved in the 3;8 translocation could alsohave value in the diagnosis of other tumors which result from othertypes of alterations to the gene involved in the 3;8 translocation.

SUMMARY OF THE INVENTION

[0011] The present invention satisfies the need identified above bydescribing the cloning and sequencing of human DNA sequences which arerearranged in the t(3;8)(p14.2; q24.1) chromosomal translocation whichoccurs in renal and thyroid carcinomas. This chromosomal translocationor rearrangement was shown to fuse sequences from a novel gene which thepresent inventors have named TRC8 (short for Translocation in RenalCancer from Chromosome 8) on chromosome 8 with the FHIT gene onchromosome 3p (the FHIT gene sequence is set forth as SEQ ID NO: 8; thecorresponding amino acid sequence is set forth as SEQ ID NO: 9). Thesequence of the novel TRC8 gene and the TRC8 protein, as well as thesequence of the t(3;8) fusion genes (5′TRC8/3′ FHIT and 5′FHIT/3′ TRC8)and the fusion proteins encoded by these fused genes are disclosedherein. A summary of certain aspects the present invention has recentlybeen published in the scientific literature (R. M. Gemmill, et al.,Prot. Natl. Acad. Sci. 95, 9572-9577 (1998)).

[0012] Identification of this gene is important because various types ofalterations or mutations of TRC8 appear to be involved with differenttypes of tumors and cancers. As just noted, the 3;8 translocation isinvolved in certain renal cancers. As described in greater detail below,a tumor-specific mutation in the 5′ untranslated region is associatedwith certain renal carcinomas. Additionally, recent work by B. T. Tehand coworkers (Genes Chromosomes Cancer 21, 260-264 (1998)) suggeststhat another rearrangement involving TRC8 (a (8;9)(q 24.1 ;q 34.3)translocation) may be associated with certain renal oncocytomas. Thus,detection of alterations in TRC8 has utility in the detection of tumorformation.

[0013] More particularly, the present invention provides an isolatedpolynucleotide molecule encoding the polypeptide comprising the aminoacid sequence of SEQ ID NO: 2. In one particular aspect, thepolynucleotide is the polynucleotide molecule of SEQ ID NO: 1, orvariants thereof. In another aspect, the polynucleotide comprisesnucleotides 238 to 2229 of SEQ ID NO: 1. The present invention furthercontemplates fragments of the polynucleotide comprising SEQ ID NO: 1that are at least 50 nucleotides, at least 100 nucleotides, at least 250nucleotides, at least 500 nucleotides and at least 1000 nucleotides inlength.

[0014] In another aspect, the present invention provides apolynucleotide sequence which hybridizes to the polynucleotide sequenceof SEQ ID NO: 1 under stringent conditions. The invention furtherprovides polynucleotide sequences comprising the complement of SEQ IDNO: 1 or variants thereof. Such complementary nucleic acid sequences mayinclude the complement of the entire sequence of SEQ ID NO: 1, orfragments thereof. More particularly, the present invention provides anisolated nucleic acid molecule comprising a nucleotide sequence selectedfrom the group consisting of: (a) a deoxyribonucleotide sequencecomplementary to nucleotides 238 to 2229 of SEQ ID NO: 1; (b) aribonucleotide sequence complementary to nucleotides 238 to 2229 of SEQID NO: 1; (c) a nucleotide sequence complementary to thedeoxyribonucleotide sequence of (a) or to the ribonucleotide sequence of(b); (d) a nucleotide sequence of at least 12 consecutive nucleotidescapable of hybridizing to nucleotides 238 to 2229 of SEQ ID NO: 1; and(e) a nucleotide sequence capable of hybridizing to a nucleotidesequence of (d).

[0015] In a yet a further embodiment, the present invention provides anisolated polynucleotide comprising at least a portion of SEQ ID NO: 1 orvariants thereof which are contained in a recombinant expression vector.The recombinant vector may be contained within a host cell in anotheraspect of the present invention. The present invention is not limited bythe particular type of host cell that can be utilized. Thus, forexample, the host cell may be a human cell, a yeast cell, a bacterialcell, etc.

[0016] The present invention also provides a method for detecting thepresence of TRC8 in a biological sample. The method comprises the stepsof: (a) selecting a probe from SEQ ID NO: 1 which specificallyhybridizes to TRC8; (b) hybridizing the probe with a biological sample;(c) detecting the presence of a hybridization complex formed by thehybridization of the probe with the TRC8 nucleic acid in the sample,wherein the presence of the complex is indicative of the presence ofTRC8 nucleic acid in the biological sample.

[0017] In a further embodiment, the present invention provides primerswhich are specific for TRC8 and which can be used in polymerase chainreaction tests to detect the gene. For example, the present inventionprovides polynucleotide molecules comprising a nucleotide sequenceselected from the group consisting of SEQ ID NO: 19 to SEQ ID NO: 45,although this is not an exhaustive list of such primers.

[0018] A method for producing a polypeptide comprising the amino acidsequence of SEQ ID NO: 2 or fragments thereof is also provided for bythe present invention. This method generally comprises: (a) culturing ahost cell which includes an expression vector containing an isolatedpolynucleotide encoding at least a fragment of the TRC8 polypeptideunder conditions suitable for the expression of the TRC8 polypeptide and(b) recovering the polypeptide from the host cell culture. The presentinvention further provides a polypeptide product of the expression in ahost cell of a DNA according to the method just described.

[0019] An isolated polynucleotide molecule including sequences locatedin the 5′ flanking region to the coding region of TRC8 (SEQ ID NO: 6) isalso provided for by the present invention. More specifically, thesesequences include nucleotides in the 5′ untranslated region, exon 1 anda portion of the coding region of TRC8. The present invention alsoincludes isolated nucleic acid molecules which are complementary to thenucleotide sequence of SEQ ID NO: 6 or fragments thereof.

[0020] In another aspect, the invention includes an isolatedpolynucleotide molecule of SEQ ID NO: 7 which occurs in certain sporadicrenal cell carcinomas. More specifically, the present invention includesan isolated polynucleotide molecule comprising nucleotides 153-176 ofSEQ ID NO: 7. The present invention further contemplates sequences whichare complementary to these sequences found in sporadic renal cellcarcinomas.

[0021] In yet a further embodiment, the present invention providesisolated polynucleotides which correspond to the two gene fusionscreated after the t(3;8)(p14.2;q24.1) translocation event, i.e. theTRC8/FHIT fusion (SEQ ID NO: 10) and the FHIT/TRC8 fusion (SEQ ID NO:11). As used herein, the TRC8/FHIT fusion or gene refers to thereciprocal fusion wherein the 5′ region of TRC8 is fused to the 3′region of FHIT; the term FHIT/TRC8 fusion or gene refers to the fusionwherein the 5′ region of FHIT is fused to the 3′ region of TRC8 (seeFIG. 1 for pictorial view of the translocation). For each of these twogene fusions, the present invention also provides an isolatedpolynucleotide sequence selected from the group consisting of: (a) adeoxyribonucleotide sequence complementary to the gene fusion, (b) aribonucleotide sequence complementary to the gene fusion; and anucleotide sequence complementary to the deoxyribonucleotide sequence of(a) or to the ribonucleotide sequence of (b).

[0022] Utilizing the sequences of the TRC8/FHIT and FHIT/TRC8 fusiongenes, the present invention provides methods of identifying thepresence of nucleic acids containing the TRC8/FHIT or FHIT/TRC8 fusions.In particular, the sequences described herein can be used to detect thegene fusions by means well-known in the art such as Southern andNorthern blots and the like, fluorescence in situ hybridization (FISH),polymerase chain reaction (PCR) amplification and other nucleic acidhybridization and detection methods.

[0023] Because, as set forth above, various alterations of TRC8 havebeen shown to be associated with at least certain renal and thyroidcarcinomas, the nucleotide sequences described herein can be used as adiagnostic for assessing renal or thyroid tumor formation in humans. Ingeneral, such diagnostic methods involve determining whether the TRC8gene has been rearranged or mutated, a rearranged or mutated TRC8 genebeing indicative of a renal or thyroid tumor. A variety of techniquescan be utilized to determine whether the TRC8 gene has been altered aswould be appreciated by those skilled in the art including PCR analysis,various amplification and hybridization methods, including for example,single-stranded conformational polymorphism (SSCP) analysis.

[0024] An example of one specific diagnostic method involvesascertaining whether there is a breakpoint in the TRC8 gene betweenbases 418 and 419 of the nucleotide sequence of SEQ ID NO: 1. Thus, forexample, nucleic acid probes which span the fusion site of the TRC8/FHITfusion (between bases 418 and 419 of SEQ ID NO: 10) or the fusion siteof the FHIT/TRC8 fusion (between bases 252-253 of SEQ ID NO: 11) can beused to detect a 3;8 chromosomal translocation by contacting the nucleicacid probe with a biological sample and then determining whether theprobe specifically hybridizes to TRC8/FHIT DNA or to FHIT/TRC8 DNA,respectively. More specifically, a method of the present invention mayinclude: (a) contacting one of the nucleic acid probes which span thefused site of the TRC8/FHIT gene fusion with a sample and (b)determining whether the probe specifically hybridizes with DNAcontaining the fused site of TRC8/FHIT but not with TRC8 DNA or FHITDNA. Similar methods can also be used with probes specific for DNAincluding the FHIT/TRC8 breakpoint.

BRIEF DESCRIPTION OF DRAWINGS

[0025]FIG. 1 illustrates a fusion of FHIT and TRC8 genes by the 3;8translocation. Normal chromosomes 3 p and 8 q are shown schematicallyalong with FHIT exons 1 through 5 (non-coding regions are shaded; codingregions are black) and TRC8 exons 1 and 2. Both genes are transcribedaway from their respective centromeres (dot on left). The 3;8 breakpoint(indicated schematically by the vertical arrow) interrupts FHIT betweenuntranslated exons 3 and 4 to generate the der (8) and der (3)chromosomes. TRC8 is interrupted between the 5′ and 3′ coding exons.Nested primers RI (SEQ ID NO: 12), R2 (SEQ ID NO: 13) and R3 (SEQ ID NO:15) within FHIT exon 4 were used for 5′ RACE (arrow heads). FHIT exon 4oligonucleotide R4 (SEQ ID NO: 17) identified bona fide RACE productswhich were tested with the exon 3 oligonucleotide F4 (SEQ ID NO: 16) toidentify putative gene fusions. The other primers indicated were usedfor RT-PCR and mapping experiments (F1—SEQ ID NO: 18; R-M—SEQ ID NO: 19;F-O—SEQ ID NO: 20; and EMR —SEQ ID NO: 21).

[0026]FIG. 2A is a schematic of TRC8 coding domains. The coding region(bold line) containing 664 amino acids extends from bp 1 to 1992(numbering of bases in this particular figure begins with the first baseof the first codon coding for the first methionine; these basescorrespond to bases 238 to 2229 of SEQ ID NO: 1) and is flanked by 5′and 3′ UTRs (thin lines). Promoters predicted from corresponding genomicsequences are indicated by horizontal arrows. Eleven predictedtransmembrane (TM) domains (light gray) and the RING-H2 motif (RingFinger, dark gray) are indicated along with the putative sterol sensingdomain and the patched homology. The 3;8 translocation breakpoint occurswithin the second transmembrane segment disrupting the sterol sensingdomain (SSD) between amino acids 60 and 61 of SEQ ID NO: 2. The positionof a mutation found in the sporadic kidney tumor RCC #1 is indicated.

[0027]FIG. 2B shows the predicted amino acid sequence of TRC8 (SEQ IDNO: 2). The sequence (also listed in GenBank as accession number3395787) begins with the first methionine present in the isolated cDNAs.The 3:8 translocation breakpoint occurs between amino acids 60 and 61.Predicted TM segments are underlined and three potential glycosylationsites are indicated by asterisks. Two regions showing similarity topatched from Drosophila melanogaster are shaded, including the SSD and aregion homologous to the second extracellular loop of patched (PTC). TheRING-H2 motif is boxed.

[0028]FIG. 2C depicts the amino acid sequence homology between TRC8 (SEQID NO: 2) and Drosophila patched (SEQ ID NO: 3). A portion of the Dm Ptcsequence (amino acids 883-978 of SEQ ID NO: 3; GenBank accession number552099 (protein), M28999 (gene)) was aligned with a portion of the TRC8amino acid sequence (residues 344 to 443 of SEQ ID NO: 2) by gappedBLAST. Identical amino acids are indicated by white letters on blackwhile similar amino acids (positive scores in a PAM250 matrix) areshaded. Two TRC8 TM segments within this homology region are underlined.

[0029]FIG. 2D shows the alignment of the amino acid sequence of humanTRC8 (SEQ ID NO: 2), human HMG-CoA reductase (SEQ ID NO: 4) and humanpatched (SEQ ID NO: 5) within the putative sterol-sensing domain (SSD).Human sequences for HMG-CoA reductase (residues 65-221 of SEQ ID NO: 4;GenBank accession number 306865; Swissprot accession number P04035) andPatched (amino acids 440 to 601 of SEQ ID NO: 5; GenBank accessionnumber 1381236) were aligned with TRC8 by gapped BLAST. Identical aminoacids are indicated by white letters on black, while similar amino acids(positive scores in a PAM250 matrix) are shaded; TM segments within theputative SSD of TRC8 are underlined.

[0030]FIG. 2E illustrates the ring-finger domain. A portion of TRC8(amino acids 547 to 585 of SEQ ID NO: 2) is shown compared to theRING-H2 consensus motif (SEQ ID NO: 46).

[0031]FIG. 3A is an analysis of TRC8 expression by a Northern blotanalysis. Gel resolved polyadenylated RNA (2 μg) from adult humantissues (Clontech Labs, Palo Alto, Calif.) was hybridized underrecommended conditions with a 1.5 kb 3 ′ TRC8 cDNA encompassing most ofthe TM segments and the ring finger (bp 83 to 1623, where bp 1 is thefirst base of the coding region; this corresponds to bp 321 to 1861 ofSEQ ID NO: 1). A second, largely non-overlapping probe (bp 1446 to 2212,where bp 1 is the first base of the coding region; this corresponds tobp 1684 to 2450 of SEQ ID NO: 1) yielded essentially the same pattern.The filter was exposed for 18 hours at −80° C.

[0032]FIG. 3B is an analysis of TRC8 expression by a dot blot analysis.A Clontech human RNA master dot blot was hybridized with the same probeas in (3A) under recommended conditions and exposed for 15 h. Final washconditions were 0.1×SSC, 0.5% SDS @ 55° C. for 20 min. Signals werecollected on a Molecular Dynamics Phosphorimager. Blank positionsincluded B8, F5-F8 and G8. Central nervous system tissues (A1-A8 andB1-B7) included (in order) whole brain, amygdala, caudate nucleus,cerebellum, cerebral cortex, frontal lobe, hippocampus, medullaoblongata, occipital lobe, putamen, substantia nigra, temporal lobe,thalamus, sub-thalamic nucleus and spinal cord. Musculature anddigestive tissues (C1-C8) included heart, aorta, skeletal muscle, colon,bladder, uterus, prostate and stomach. Secretory tissues (D1-D8)included testis, ovary, pancreas, pituitary, adrenal, thyroid, salivaryand mammary glands. Miscellaneous tissues (E1-E8 and F1-F4) includedkidney, liver, small intestine, spleen, thymus, peripheral leukocytes,lymph node, bone marrow, appendix, lung, trachea and placenta. Fetaltissues (G1-G7) included brain, heart, kidney, liver, spleen, thymus andlung. All control spots (yeast and E. coli RNAs, human Cotl and totalhuman DNAs) were blank (not shown).

[0033]FIG. 4A illustrates the localization of 5′ TRC8 sequences tochromosome 8q. Primers R-M (SEQ ID NO: 19) and F-O (SEQ ID NO: 20) (seeFIG. 1 for general location of TRC8 to which primers hybridize) amplifyan 82 bp fragment specific for the 5′ portion of TRC8. Templates inlanes 1 through 11 included, respectively, AG4103 (normal human), CHOglyA (hamster), UCTP-2A3 (chromosome 3 only hybrid), 706-B6, clone 17(chromosome 8 only hybrid), TL12-8 [t(3;8) der(3) hybrid], 3;8/4-1[t(3;8) der(8) hybrid], YAC 880A9 (chromosome 8-specific YAC spanning3;8 breakpoint), YAC 850A6 (chromosome 3-specific YAC spanning 3;8breakpoint), HD-7 (genomic phage clone carrying the 3;8 breakpointregion from chromosome 8), 2A7 (longest 5′ RACE clone), water control.Molecular size standards are indicated in base pairs.

[0034]FIG. 4B is a Southern blot in which the same hybrid and YAC DNAslisted in FIG. 4A were digested with EcoRI and then Southern blotted.The filter was hybridized with a 1.4 kb TRC8 cDNA fragment which derivesfrom the 3′ end. The normal human TRC8 fragment is >15 kb which isreduced to ˜12 kb by the translocation (arrow). The cross-hybridizingfragment in hamster DNA (lanes 2-6) is 8 kb.

[0035]FIG. 5A shows an RT-PCR analysis of fusion product expression.RNAs isolated from the t(3;8) lymphoblastoid cell line TL9944 (R. M.Gemmill, et al., Genomics 4, 28 (1989)) and from a control breastcarcinoma cell line HTB 121 were treated with or without reversetranscriptase, as indicated (+ or −) and analyzed for expression of FHITand TRC8 by PCR. Four primers specific for 5′ and 3′ portions of eachgene, Fl (SEQ D NO: 18) and RI (SEQ D NO: 11 for FHIT and R-M (SEQ D NO:19) and EMR (SEQ ID NO: 21) for TRC8 (see FIG. 1 for general section ofgene to which primers hybridize), were used in combination to detectboth wild-type and putative chimeric transcripts. The FHIT primer pairgenerated a product of the expected size (231 bp), as did the TRC8primer pair (651 bp). Reciprocal chimeric products were amplified usingR-M (SEQ ID NO: 19) plus R1 (SEQ ID NO: 12) for 5′ TRC8/3′ FHIT, and F1(SEQ ID NO: 18) plus EMR (SEQ ID NO: 21) for 5′ FHIT/3′ TRC8. Predictedsizes of the chimeric products are 188 and 694 bp, respectively. Lanes1-16 are in order from left to right.

[0036]FIG. 5B lists the sequences of 3;8 chimeric transcripts. TheRT-PCR amplified cDNAs in lanes 11 and 15, corresponding to thereciprocal chimeric transcripts, were purified and sequenced on bothstrands. Bases surrounding the boundary between FHIT exons 3 and 4 areshown with FHIT sequences italicized (bases 399 to 438 of SEQ ID NO: 10(5′TRC8/FHIT3′ fusion) and bases 234 to 272 of SEQ ID NO: 11(5′FHIT/TRC83′ fusion). The precise position of the fusion on both TRC8and FHIT transcripts is indicated. (For TRC8, the base numbering in FIG.5B assumes that the first base in the coding region is base number 1.Thus, bp 180 of TRC8 is bp 418 of SEQ ID NO: 1. For FHIT, bp 137corresponds to bp 253 of SEQ ID NO: 8.)

[0037]FIG. 6A illustrates the detection of a tumor-specific somaticmutation by Single Stranded Conformational Polymorphism Analysis (SSCP)and heteroduplex analysis. DNA samples were PCR amplified using primersflanking the first coding exon of TRC8 (M. Le Beau, et al. GenesChromosomes Cancer 21, 281 (1998)). The products were denatured,separated on a non-denaturing MDE gel and detected by silver staining.Samples included matched tumor and normal DNAs from patients 1 and 7(lanes 5-8, respectively) and an unrelated normal control (AG4103, lane9). A separate SSCP gel was used to isolate four individual SSCP bandsfrom RCC #1 (lane 5, marked by an arrow or arrow heads). The excisedbands A to D corresponded to the indicated bands in lane 5 from top tobottom. These last templates were re-amplified and analyzed by SSCP todetermine if they contained mutant or wild-type sequences. Comparison tolane 5 suggested that bands A and C contained primarily mutant DNA, bandB was a mixture of mutant and wild-type and band D was wild-type only.The top and bottom panels show the SSCP and heteroduplex results,respectively.

[0038]FIG. 6B illustrates that Renal Cell Carcinoma (RCC)#1 contains a12 bp duplication in the 5′ UTR. Purified PCR products shown in (A)(lanes 1 through 4) were sequenced. The mutation consisted of a 12 bpdirect duplication (underlined) at bp position -73 (as numbered when thefirst base of the coding region is bp 1; this corresponds to bp 165 ofSEQ ID NO: 1.) which was present in the tumor sample but not thecorresponding normal DNA. The repeat is from bp 165 to 176 of SEQ ID NO:7.

[0039]FIG. 7 demonstrates that TRC8 is amplified in a sub-set of variantSmall Cell Lung Carcinomas (SCLCs). A Southern blot of EcoRI digestedtumor cell line DNAs was prepared with nearly identical amounts of DNA(2 Tg) loaded in each lane, as determined by ethidium bromidefluorescence (bottom panel). The cell lines included 8 cervicalcarcinomas (ME180, SiHa, HeLa, CC19, Caski, MS751, C33A and C41) and 7small cell lung carcinomas of the variant sub-type (H82, H196, H211,H360, H433, H437 and H524), as indicated. The filter was hybridizedsequentially with probes for TRC8 (top panel), a control locus on 3q (MJ1536, second panel) and a genomic probe (380j9) which derives fromwithin the cMYC locus (third panel). The autoradiogram generated by TRC8was densitometrically scanned and band intensities were normalized bycomparison to the control (lane 1). TRC8 is amplified 6-fold over normalin line H211 (lane 12).

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0040] The present invention is based upon the identification andcharacterization of several new nucleic acid sequences: (a) the novelgene TRC8, which encodes a novel TRC8 protein, (b) a sequence includingbases in the 5′ flanking region to the TRC8 coding region plus exon 1,(c) a nucleotide sequence for a mutated TRC8 which was found in asporadic renal cell carcinoma, (d) the 5′ TRC8/3′ FHIT fusion and therelated 5′ FHIT/3′ TRC8 fusion, which result from a chromosomaltranslocation event (specifically the t(3;8)(p14.2;q24.1)), thischromosomal translocation being associated with human renal and thyroidtumors in the 3;8 family.

[0041] TRC8 appears to be a critical gene in the 3;8 translocation andappears to be linked with various tumors and cancers based on thefollowing: (i) its similarity to patched, which in turn is responsiblefor the hereditary basal cell carcinoma syndrome (H. Hahan, et al., Cell85, 841-851 (1996); R. L. Johnson, et al., Science 272, 1668-1671(1996)), (ii) the preservation and expression of FHIT coding sequencesin 3;8 translocation containing cells (in contrast to the disruption ofTRC8 coding sequences), and (iii) its demonstrated mutation in asporadic renal carcinoma Furthermore, recent work by other researchershas provided cytogenetic evidence for another set of renal tumors thatappear to be associated with alterations in TRC8, including a 8q24.1breakpoint as in the 3;8 translocation described herein. Morespecifically, analysis of the lymphocytes of a patient suffering frombilateral multifocal renal oncocytomas and cysts showed a constitutionalreciprocal t (8;9) (q24.1; q 34.3) Teh, et al. (Genes Chromosomes Cancer21, 260-264 (1998)). The fact that several different alterations of TRC8is associated with various tumors and cancers indicates that thedetection of alterations in TRC8 has utility in the diagnosis of certaintumors and cancers. The type of alterations could include translocationssuch as the t(3;8), as well as substitutions, deletions, insertions,inversions, etc.

[0042] One embodiment of the present invention provides an isolatednucleic acid sequence, TRC8 (SEQ ID NO: 1), which encodes the TRC8protein (SEQ ID NO: 2). As described in further detail below, TRC8encodes a predicted 664-aa, multitransmembrane protein with similarityto patched from Drosophila melanogaster. This similarity includes thesecond extracellular domain of patched, which is involved in bindingsonic hedgehog, as well as its putative sterol-sensing domain (SSD). Inaddition, the first 480 amino acids of TRC8 and amino acids 440-1100 ofpatched share an organization similarity. This similarity begins withthe common SSD, followed by the divergent region that is nonconservedamong patched homologues (J. Motoyama, et al., Nat. Genet. 18, 104-106(1998)), and finally by the conserved second extracellular loop. TRC8lacks the first extracellular loop of patched and likewise shows nosimilarity after the second extracellular loop. Therefore, although TRC8has similarity to patched and is predicted to be a plasma membraneprotein by PSORT (K. Nakai, and M. Kanehisa, Genomics 14, 897-911(1992)), it is not the type of direct homologue as is the Patched 2gene, for instance (J. Motoyama, et al., Nat. Genet. 18, 104-106(1998)).

[0043] As those skilled in the art will appreciate, a number ofdifferent nucleotide sequences can encode for the TRC8 protein becauseof the degeneracy of the genetic code. Consequently, the presentinvention contemplates each and every possible variation of thenucleotide sequence that can be made by selecting from the possiblecodon choices for a given amino acid in the TRC8 protein sequence. Onesuch sequence is the sequence listed as SEQ ID NO: 1. More particularly,the present invention includes an isolated polynucleotide comprisingnucleotides 238 to 2229 of SEQ ID NO: 1 (the coding region of the TRC8gene; the stop codon includes bases 2230 to 2232 of SEQ ID NO: 1). Thepresent invention also includes the production by synthetic chemistry ofDNA sequences, or fragments thereof, which encode for the TRC8 proteinand the subsequent insertion of such synthetic sequences into any of thenumber of currently available expression vectors and cell systems whichare known to those skilled in the art.

[0044] In another embodiment, the present invention provides an isolatednucleic acid molecule comprising a nucleotide sequence selected from thegroup consisting of: (a) a deoxyribonucleotide sequence complementary tonucleotides 238 to 2229 of SEQ ID NO: 1; (b) a ribonucleotide sequencecomplementary to nucleotides 238 to 2229 of SEQ ID NO: 1; (c) anucleotide sequence complementary to the deoxyribonucleotide sequence of(a) or to the ribonucleotide sequence of (b); (d) a nucleotide sequenceof at least 12 consecutive nucleotides capable of hybridizing tonucleotides 238 to 2229 of SEQ ID NO: 1; and (e) a nucleotide sequencecapable of hybridizing to a nucleotide sequence of (d).

[0045] By inserting the TRC8 nucleic acid sequence (i.e., SEQ ID NO: 1)into an appropriate vector, it is possible to prepare large quantitiesof the TRC8 sequence using methods which are well-known to those withskill in the art. Alternatively, the TRC8 nucleic acid sequence can beinserted into an expression vector and the vector placed in a host cellto produce the TRC8 protein (i.e. SEQ ID NO: 2). The TRC8 protein canthen be isolated from the host cell culture according to standardpurification techniques. A number of host/vector systems are availablefor the amplification of the TRC8 nucleic acid sequence and/or theprotein expressed by the TRC8 gene. Such systems include, but are notlimited to, plasmid and viral vectors, and eukaryotic and procaryotichosts.

[0046] The present invention also provides methods for detecting thepresence of TRC8 in a biological sample. One method comprises the stepsof: (a) selecting a probe from SEQ ID NO: 1 which specificallyhybridizes to TRC8; (b) hybridizing the probe with a biological sample;and (c) detecting the presence of a hybridization complex formed by thehybridization of the probe with the TRC8 nucleic acid in the sample,wherein the presence of the complex is indicative of the presence ofTRC8 nucleic acid in the biological sample. Another method includescontacting a nucleic acid probe which is at least 12 continuousnucleotides in length and is specific for binding to human TRC8 genewith the biological sample under conditions which allow the nucleic acidprobe to anneal to complementary sequences in the sample and thendetecting duplex formation between the nucleic acid probe and thecomplementary sequences. The nucleic acid probe used may be asubsequence of the entire human TRC8 gene.

[0047] In a further embodiment, the present invention provides primerswhich are specific for TRC8. These primers can be used to amplify TRC8and thus detect its presence according to PCR methodologies which arewell-known in the art. In particular, the present invention providespolynucleotide molecules comprising a nucleotide sequence selected fromthe group consisting of SEQ ID NO: 19 to SEQ ID NO: 45, inclusively.

[0048] A method for producing a polypeptide comprising the amino acidsequence of SEQ ID NO: 2 or fragments thereof is also provided for bythe present invention. This method generally comprises: (a) culturing ahost cell which includes an expression vector containing an isolatedpolynucleotide encoding at least a fragment of the TRC8 polypeptideunder conditions suitable for the expression of the TRC8 polypeptide and(b) recovering the polypeptide from the host cell culture. The presentinvention further provides a polypeptide product in a host cell of a DNAaccording to the method just described.

[0049] An isolated polynucleotide molecule including sequences locatedin the 5′ flanking region to the coding region of TRC8 (SEQ ID NO: 6) isalso provided for by the present invention. More specifically, thesesequences include exon 1 and a portion of the coding region of TRC8. Thepresent invention also includes isolated nucleic acid molecules selectedfrom the group consisting of: (a) a deoxyribonucleotide sequencecomplementary to SEQ ID NO: 6; (b) a ribonucleotide sequencecomplementary to SEQ ID NO: 6; (c) a nucleotide sequence complementaryto the deoxyribonucleotide sequence of (a) or to the ribonucleotidesequence of (b); (d) a nucleotide sequence of at least 12 consecutivenucleotides capable of hybridizing to nucleotides of SEQ ID NO: 6; and(e) a nucleotide sequence capable of hybridizing to a nucleotidesequence of (d).

[0050] In another aspect, the invention includes an isolatedpolynucleotide molecule of SEQ ID NO: 7 which occurs in certain sporadicrenal cell carcinomas. More specifically, the present invention includesan isolated polynucleotide molecule comprising nucleotides 153-176 ofSEQ ID NO: 7. In this regard, the present invention further provides anisolated nucleic acid molecule selected from the group consisting of:(a) a deoxyribonucleotide sequence complementary to nucleotides 153-176of SEQ ID NO: 7; (b) a ribonucleotide sequence complementary tonucleotides 153-176 of SEQ ID NO: 7; and (c) a nucleotide sequencecomplementary to the deoxyribonucleotide sequence of (a) or to theribonucleotide sequence of (b).

[0051] In another aspect, the present invention provides isolatedpolynucleotide sequences which correspond to the two gene fusionscreated after the t(3;8)(p14.2;q24.1) translocation event, i.e. theTRC8/FHIT fusion (SEQ ID NO: 10) and the FHIT/TRC8 fusion (SEQ ID NO:11). As noted above, the TRC8/FHIT fusion refers to the fusion whereinthe 5′ region of TRC8 is fused to the 3′ region of FHIT; the termFHIT/TRC8 fusion refers to the fusion wherein the 5′ region of FHIT isfused to the 3′ region of TRC8. For each of these two gene fusions, thepresent invention also provides an isolated polynucleotide sequenceselected from the group consisting of: (a) a deoxyribonucleotidesequence complementary to the gene fusion, (b) a ribonucleotide sequencecomplementary to the gene fusion; and (c) a nucleotide sequencecomplementary to the deoxyribonucleotide sequence of (a) or to theribonucleotide sequence of (b).

[0052] The present invention provides nucleic acid probes selected fromthe group consisting of: (a) a deoxyribonucleotide sequence which is aDNA fragment comprising contiguous nucleotides on the 5′ and 3′ sides ofthe fused site of either TRC8/FHIT fused DNA or FHIT/TRC8 fused DNA, (b)a ribonucleotide sequence complementary to the deoxyribonucleotidesequence of (a), and a nucleotide sequence complementary to thedeoxyribonucleotide sequence of (a) or to the ribonucleotide sequence of(b), wherein the fused site is between bases 418 and 419 of thenucleotide sequence of SEQ ID NO: 0 for the TRC8/FHIT fusion and betweenbases 252 and 253 of SEQ ID NO: 11 for the FHIT/TRC8 fusion.

[0053] In another embodiment, the present invention provides a pair ofoligionucleotides which can be used in PCR analysis for example, whereinone of the oligonucleotides specifically hybridizes with the TRC8-FHITfused DNA comprising the contiguous nucleotide sequence of SEQ ID NO: 10on the 3′ side of the fused site and the other oligonucleotidespecifically hybridizes with the TRC8-FHIT fused DNA on the 5′ side ofthe fused site, the fused site being located between bases 418 and 419of SEQ ID NO: 10. Similarly, the present invention also provides a pairof oligonucleotides wherein one of the oligonucleotides specificallyhybridizes with the FHIT/TRC8 fused DNA comprising the contiguousnucleotide sequence of SEQ ID NO: 11 on the 3′ side of a fused site andthe other oligonucleotide specifically hybridizes with the FHIT/TRC8fused DNA on the 5′ side of said fused site, said fused site beinglocated between bases 252 and 253 of SEQ ID NO: 11.

[0054] Because alterations of the TRC8 gene have been implicated withseveral different renal and thyroid carcinomas, the nucleotide sequencesdescribed herein can be used as a diagnostic for assessing renal orthyroid tumor formation in humans. In general, such diagnostic methodsinvolve determining whether the TRC8 gene has been rearranged ormutated, a rearranged or mutated TRC8 gene being indicative of a renalor thyroid tumor. The mutations may be of various types including, forexample, deletions, substitutions and inversions

[0055] Various methods which are well known to those skilled in the artcan be used to identify alterations in TRC8. One method, for example,involves using paired primer sets to amplify DNA samples. Alterationssuch as mutations can then be identified using single-strandedconformational polymorphism analysis (SSCP). Examples of the types ofprimers which could be utilized and additional specifics regarding theSSCP methodology is set forth more fully in Example 5 below.

[0056] Although the 3;8 translocation is but one of what appears to beseveral alterations to TRC8 which is associated with different tumorsand cancers, the following methods illustrate how the 3;8 translocationcan be detected. It is important to recognize, however, that similarmethods could be used to detect other chances to TRC8.

[0057] One example of an assay method which can be utilized to detectthe 3;8 translocation and the formation of the TRC8/FHIT and FHIT/TRC8fusions involves selective amplification of sequences within a samplewhich contains the TRC8/FHIT and FHIT/TRC8 polynucleotides (SEQ ID NO:10 and SEQ ID NO: 11, respectively). The present invention also includesmethods which identify nucleic acids containing the TRC8/FHIT andFHIT/TRC8 fusions but which do not require sequence amplification fordetection. Such methods include Southern and Northern blot hybridizationtests and fluorescence in situ hybridization (FISH) of chromosomalmaterial, using probes derived from the nucleic acids of the presentinvention.

[0058] As noted above, the nucleic acid probes of the present inventioncan be DNA or RNA probes. Such probes can be prepared according tomethods which are known in the art (see for example, Molecular Cloning,(Sambrook, et al., Eds.), Cold Spring Harbor Press, Cold Spring Harbor,N.Y. (1989)). One with skill in the art can employ the techniques suchas described in the preceding reference and the sequences describedherein, or fragments thereof, as probes.

[0059] The detection methods of the present invention can be utilizedwith a variety of sample types. A non-exhaustive list of the type ofsamples that can be used include cells or tissues, extracts of cells ortissues containing protein, membranes, nucleic acids or combinationsthereof, and biological fluids such as blood, serum and plasma. Methodsfor preparing such extracts are known in the art and can readily beadapted by one skilled in the art to obtain a sample which isappropriate for the type of detection test being conducted (see, forexample, K. Budelier et al., Chapter 2, “Preparation and Analysis ofDNA,” M. E. Greenberg, et al., Chapter 4, “Preparation and Analysis ofRNA,” and M. 5 Moos, et al., Chapter 10, “Analysis of Proteins,” inCurrent Protocols in Molecular Biology, Wiley Press, Boston, Mass.(1993)).

[0060] More specifically, one diagnostic method involves ascertainingwhether there is a breakpoint in the TRC8 gene between bases 418 and 419of the nucleotide sequence of SEQ ID NO: 1. For example, the nucleicacid probes described above which span the fused site of the TRC8/FHITfusion or the FHIT/TRC8 fusion can be used to detect a 3;8 chromosomaltranslocation by contacting the nucleic acid probe with a biologicalsample and then determining whether the probe specifically hybridizes toTRC8/FHIT DNA or to FHIT/TRC8 DNA. Of course probes could also be usedto probe for complementary DNA or RNA sequences to the two fusedsequences. Thus, for example, a method of the present invention mayinclude: (a) contacting one of the nucleic acid probes which span thebreakpoint of the TRC8/FHIT gene fusion with a sample and (b)determining whether the probe specifically hybridizes with DNAcontaining the fused site of TRC8/FHIT but not with TRC8 DNA or FHITDNA. Similar methods can also be used with probes specific for DNAincluding the FHIT/TRC8 breakpoint. A second method would directlydetect mutations in TRC8.

[0061] The following specific examples further describe the presentinvention and further illustrate the features and advantages provided bythe present invention.

EXAMPLE 1 Identification and Sequence Analysis of TRC8

[0062] (a) Cell Lines and Genomic Clones

[0063] Tumor cell lines were obtained from American Type CultureCollection (Gaithersburg, Md.), except for somatic cell hybrids whichwere generated by the present inventors previously and reported by H. A.Drabkin, et al. (Cancer Cells 7, 63 (1989)). The hybrids TL12-8 and3;8/4-1 contain the der (3) and der (8) chromosomes, respectively, fromthe t(3;8) lymphoblastoid cell line TL9944 (without either a normal 3 or8 chromosome). The human lymphoblastoid line AG4103 served as a normalcontrol. Isolation of DNA and RNA was performed using standard methods.

[0064] The HD-7 genomic phage clone carrying the 3;8 translocationbreakpoint from the der(8) chromosome was isolated from a libraryprepared from the TL9944 cell line in λ FHXII (Stratagene, Inc., LaJolla, Calif.). A chromosome 3 probe (λ4040) which maps just distal tothe 3;8 breakpoint was used for screening (F. L. Boldog, et al., Proc.Natl. Acad. Sci. USA 90, 8509 (1993)).

[0065] (b) 5′ Race

[0066] RNA was isolated from TL9944 lymphoblastoid cells carrying the3;8 translocation (see, R. M. Gemmill, et al., Genomics. 4, 28 (1989)),and then subjected to RACE (see, M. A. Frohman, PCR. Methods Appl. 4,S40 (1994)). 5′ RACE was performed essentially as described by M. A.Frohman (Methods. Enzymol 218, 340 (1993)). First strand cDNA synthesisused FHIT exon 4-specific primer R1 (5′-TCAGAAGACTGCTACCTCTTCG-3′—SEQ IDNO: 12) followed by dCTP tailing with terminal deoxynucleotidyltransferase. Primary amplification utilized the AAP 5′-RACE primer froma 5′ RACE Kit sold by Gibco-BRL/Life Technologies, together with R2, anested FHIT exon 4-specific primer (5′-TCAGTGGCAGGATGCACAG-3′—SEQ ID NO:13). Second round nested PCR utilized primer AUAP (also from the 5′ RACEKit sold by Gibco-BRL/Life Technologies) with R3, a second nested FHITexon 4 - specific primer (5′-GGTCTAAGCAGGCAGGTATTC-3′—SEQ ID NO: 15).Products were cloned into a T-vector (pBluescript II K/S) analyzed byhybridization with additional internal FHIT oligonucleotides F4(5′-TGGAAGGGAGAGAAAGAG-3′—SEQ ID NO: 16) and R4(5′-GGTATTCCTAGGATAC-3′—SEQ ID NO: 17) and sequenced.

[0067] Because the t(3;8) breakpoint interrupts FHIT between exons 3 and4, 5′-RACE products were generated using nested primers within FHIT exon4 as shown in FIG. 1. Cloned amplification products were identified byhybridization with oligonucleotide R4 (SEQ ID NO: 17). Nearly 80% of R4positive clones were negative for an exon 3 oligonucleotide (F4)suggesting they might represent a gene fusion. Nine of 12 sequencedclones contained an identical novel sequence spliced exactly to the 5′end of FHIT exon 4. Mapping studies confirmed that the new sequenceswere derived from chromosome 8 (see below). As noted earlier, thepresent inventors refer to this gene as TRC8 for Translocation in RenalCancer from chromosome 8.

[0068] (c) DNA Sequence Analysis

[0069] The coding region of TRC8 was determined from multiple cDNAclones and PCR products isolated from a human fetal brain library(Stratagene, Inc.) as well as IMAGE clone 331H8 identified from dbEST.Sequencing was performed on an AB1377 through the Colorado Cancer CenterDNA Sequencing Core. Analysis for transmembrane segments was performedusing five prediction programs, including PHD_htm at EMBL(http://www.emblheidelberg.de/predictprotein/), TMpred at ISREC(http://ulrec3.unil.ch/software/ TMPRED_form.html), SOSUI at TokyoUniversity (http://www.tuat.ac.jp/˜mitaku/adv_sosui/), DAS at StockholmUniversity (http://www.biokemi.su.se/˜server/DAS/), and PSORT at OsakaUniversity (http://psort.nibb.ac.jp/). All ten transmembrane segmentsthat are underlined in FIG. 2B were predicted by at least four out ofthe five programs, although in most cases the programs did not agree onthe precise boundaries of the segment.

[0070] The DNA sequence (SEQ ID NO: 1; Genbank AF064801) contains apredicted 1992 bp open reading frame (bases 238 to 2229 of SEQ ID NO: 1,or bases 1 to 1992 if the first base in the coding region is consideredthe first base) as shown in FIG. 2A. Upstream cDNA as well ascorresponding genomic sequences are GC-rich indicative of a CpG-island.Using a promoter prediction program (http://www-hgc.lbI.gov/projects/promoter.html), four transcriptional start sites arelocated at −622, −55, −36 and −22 bp of the first methionine (thenumbering in this instance assumes that the first bp of the codingregion is base number 1; this corresponds to bp 238 of SEQ ID NO: 1).The −22 site corresponded precisely to two of the nine sequenced RACEproducts. Use of the −622 site is suggested by the longest presentlyavailable cDNA which extends to position −286 and RT-PCR experimentshave confirmed transcription to at least position −547.

[0071] The ORF is predicted to encode a 664 amino acid protein (SEQ IDNO: 2) of 76 kDa (see FIGS. 2A and 2B) with at least ten membranespanning segments. TRC8 contains two regions of similarity with the genepatched from Drosophila (the amino and sequence encoded by patched islisted as SEQ ID NO: 3), the receptor for Sonic Hedgehog (SHH) (V.Marigo, et al., Nature 384, 176 (1996); and D. M. Stone, et al., Nature384, 129 (1996)). The region from amino acids 344 to 443 of TRC8 (SEQ IDNO: 2) shows the strongest match with 60% similarity and 23% identity toamino acids 883-979 of patched (residues 883 to 979 of SEQ ID NO: 3)(See FIG. 2C); this region of patched represents most of the secondpredicted extracellular domain involved in the binding of SHH. A secondregion of patched similarity involves amino acids 22 to 179 of SEQ IDNO: 2 and encodes a putative sterol sensing domain (SSD). Such domains,identified in HMG-CoA reductase and the sterol regulatory elementbinding protein (SREBP) cleavage activating protein (SCAP), consist offive membrane spanning segments arranged with a specific spacing pattern(X. Hua, et al., Cell 87, 415 (1996)). Patched contains a putative SSD,of unknown function, from amino acids 440 to 601 of SEQ ID NO: 3 (E. D.Carstea, et al., Sci. 277, 228 (1997)). This region is 53% similar/17%identical to the SSD of HMG-CoA reductase (amino acids 65 to 221 of SEQID NO: 4), as reported by D. J. Chin, et al. (Nature 308, 613 (1984));the corresponding region from TRC8 (amino acids 22 to 179 of SEQ ID No:2) shows 63% similarity/17% identity (See FIG. 2D). Not wishing to bebound by any particular theory, the present inventors surmise, basedupon the multiple transmembrane segments and regions of patchedsimilarity, that TRC8 encodes a membrane bound receptor.

[0072] In addition, a perfect match with a ring-finger motif of theRING-H2 sub-type (SEQ ID NO: 46) (P. S. Freemont, Ann. N. YA cad. Sci.684, 174 (1993)) was found in TRC8 between amino acids 547 to 585 of SEQID NO: 2 as shown in FIG. 2E. The RING-H2 motif in TRC8 differs from thestandard RING-finger by replacement of the fourth cysteine with ahistidine. Functionally, RING-H2 motifs have been suggested to beprotein-protein or protein-lipid interaction domains. That TRC8 ishighly conserved, at least among mammals, is evident from two murineESTs (dbEST clones mu78h12 and v143c01) found to be 93% and 89%identical at the nucleotide level over 971 bp.

EXAMPLE 2 Expression of TRC8

[0073] Hybridization of TRC8 to a Northern blot (CLONETECH) preparedfrom adult human tissues and placenta identified a message ofapproximately 3.0 kb (FIG. 3A). Although the longest cDNA clones total2.5 kb, use of the −622 promoter (numbered as though the first base ofthe coding region is bp 1, i.e., base 238 of SEQ ID NO: 1), as discussedabove, would result in a 2.9-kb message, close to the observed size.Although expression in the lung and kidney appeared reduced,hybridization with a control glyceraldehyde-3-phosphate dehydrogenaseprobe (data not shown) indicated that there was less RNA present inthese samples. A human RNA dot blot revealed TRC8 message in all tissuesexamined (as shown in FIG. 3B), with the highest levels in testis (D1)and placenta (F4) and adrenal (D5); the lowest level was in thymus (G6).TRC8 is expressed in both fetal (G3) and adult kidney (E1) and in adultthyroid (D6), the suspected target organs for TRC8 aberrations in the3;8 translocation family.

EXAMPLE3 Mapping of TRC8

[0074] TRC8 sequences were localized to the immediate region of thebreakpoint on chromosome 8 by both PCR and Southern blot analysis ofhybrids, “YACs” (yeast artificial chromosomes) and phage clones (SeeFIG. 4). PCR mapping used TRC8 specific primers R-M(5′-GCCCTGCCTTTACATCATCGAC-3′—SEQ ID NO: 19) and F-O(5′-AGATCTGGAGCACGATGCAGAAC-3′—SEQ ID NO: 20) which lie within a GC richsegment. PCR reactions were performed under touch-down annealingconditions with Perkin-Elmer Buffer II and Promega AmpliTaq DNApolymerase. Touch-down annealing temperatures started at 70° C. andended at 60° C. (ΔT of −0.5° C. per cycle) for 20 cycles, followed by 15cycles at 60° C. Products generated from 10 to 40 ng of template wereseparated on a 2.0% agarose gel. cDNA synthesis utilized random hexamerprimers along with Superscript II (Life Technologies Inc., Gaithersburg,Md.). Subsequent PCR reactions were performed as above, excepttouch-down annealing temperatures were adjusted to 65° C.-55° C. The EMRprimer, specific for the 3′ portion of TRC8 was5′-TCTTGTTAGCCAAAAGACTCG-3′ (SEQ ID NO: 21), whereas the F1 primerspecific for FHIT exon 1 was 5′-TCCCTCTGCCTTTCATTCC-3′ (SEQ ID NO: 18).

[0075] Primers derived from the 5′ coding portion of TRC8 yielded theexpected product in the chromosome 8 only hybrid (lane 4) but not in achromosome 3 only hybrid (lane 3). The same product was also present onthe der(8), but not the der(3) chromosome from the 3;8 translocation(lanes 6 and 5, respectively). Similarly, the 8q24 YAC 880A9 (lane 7)was positive, as was a lambda clone, HD7 (lane 9), which contained bothchromosome 8 and 3 material from the breakpoint junction. As notedabove, the HD-7 genomic phage clone carrying the 3;8 translocationbreakpoint (in particular, the breakpoint region from the der(8)chromosome) was isolated from a library prepared from the TL9944 cellline in λFHXII (Stratagene, Inc., La Jolla, Calif.). A chromosome 3probe (λ4040) which maps just distal to the 3;8 breakpoint was used forscreening. Thus, the 5′ coding region of TRC8 is proximal to the 8q24breakpoint.

[0076] Southern blot analysis (See FIG. 4B) was used to demonstrate thatthe remaining 3′ portion was contained on the der(3) chromosome (lane5). Importantly, the probe hybridized to an altered band (arrow) in theder(3) hybrid consistent with the t(3;8) rearrangement. Together, thesedata indicate that TRC8 is localized to 8q24, is interrupted by the 3; 8translocation and that its 5′ to 3′ orientation is centromere totelomere (FIG. 1).

EXAMPLE 4 Expression of Both Reciprocal Products in (3;8) LymphoblastoidCells

[0077] To determine if both reciprocal products were expressed,RT(reverse transcriptase)-PCR analysis was performed on RNA isolatedfrom TL9944 lymphoblastoid cells carrying the 3;8 translocation. Primerswhich flanked the breakpoint and were specific for the 5′ and 3′portions of either TRC8 or FHIT (see FIG. 1) were used to demonstrateexpression of both wild-type and fusion transcripts in TL9944 cells. Ascan be seen in FIG. 5, primers specific for wild-type FHIT and TRC8generated bands of the expected size from both t(3;8) and control RNAs(lanes 1, 3, 5 and 7). In contrast, the primer pair R-M (5′-TRC8; SEQ IDNO: 19) plus RI (3′-FHIT; SEQ ID NO: 12) produced a product only fromthe translocation cell line (lane 11) as did the primer pair F1(5′-FHIT; SEQ ID NO: 18) plus EMR (3′-TRC8; SEQ ID NO: 21), lane 15. Noproducts were observed in the absence of reverse transcriptase (evenlanes). Sequence analysis (FIG. 5B) confirmed that the product from theder(8), 5′-TRC8/3′-FHIT, contained TRC8 sequences fused to FHIT exon 4.Similarly, the reciprocal product, 5′-FHIT/3′-TRC8, consisted of FHITexon 3 fused to 3′ TRC8 sequences. Thus, while FHIT is interrupted inits 5′ untranslated region, its coding sequences are contained in theder(8) product. In contrast, TRC8 is interrupted within its codingsequence and, more specifically, within the predicted sterol sensingdomain. Of note, a mutation in the sterol-sensing domain of SCAPenhances its activity and renders the molecule non-responsive toregulation by sterols (X. Hua, et al., Cell 87, 415 (1996)).

EXAMPLE 5 Identification of Tumor-Specific Mutation in TRC8 in SporadicRenal Cell Carcinomas

[0078] Single-stranded conformational polymorphism (SSCP) was performedusing twelve primer pairs covering the coding sequence and the 5′untranslated region in 32 renal carcinomas. The SSCP analysis wasperformed by the method of Spritz et al. (R. A. Spritz, et al., Am. JHum. Genet. 51 1058-1065 (1992)). Nine primer sets (sets 1-9) weredesigned to amplify the entire coding region in segments averaging 325bp and that also would span any intron-exon boundaries (see Table 1). Inaddition, three primer sets (Set P1-P3) were designed to amplify the 5′untranslated region (see Table 1). TABLE I PRIMER PRIMER SET NAMESEQUENCE SEQ ID NO: Primers Specific for TRC8 Coding Region Set 1 Set IFAGTTGCCCGCCTTAGCC SEQ ID NO:22 Set 1 Set 1R CCAAAGACACATACTCGACCC SEQ IDNO:23 Set 2 Set 2F CATAACTCTTAGTGGGGAAACATTC SEQ ID NO:24 Set 2 Set 2RTGTAACGTATCCAATTCCAAATG SEQ ID NO:25 Set 3 Set 3F TGGCACTTATCGTTCTACAGCSEQ ID NO:26 Set 3 Set 3R TCTTGTTAGCCAAAAGACTCG SEQ ID NO:27 Set 4 Set4F AGTGTTTGTCCTGGCAGTG SEQ ID NO:28 Set 4 Set 4R ACAGTTAGTGTAGAATCGCACCCSEQ ID NO:29 Set 5 Set 5F TGGCAAATGAAACTGATTCC SEQ ID NO:30 Set 5 Set 5RCATGGATAAAATGCAGGACTG SEQ ID NO:31 Set 6 Set 6F AAGACCAGAAGAGAGACTTATTCGSEQ ID NO:32 Set 6 Set 6R TGCTGTAACTGCAAACAACC SEQ ID NO:33 Set 7 Set 7FTCTTTGGCATCACTATGCAC SEQ ID NO:34 Set 7 Set 7R CTTCACAGCAGTCCTACGATTCSEQ ID NO:35 Set 8 Set 8F CCAAAAATGGCTGGAAGAC SEQ ID NO:36 Set 8 Set 8RTGTCAGATTCAGCAGCAGC SEQ ID NO:37 Set 9 Set 9F CCACCCAATGAAACTCCAG SEQ IDNO:38 Set 9 Set 9R AGTAGCACATCACAGTAAACGG SEQ ID NO:39 Primers specificfor 5′ Untranslated Region of TRC8: Set P1 Set P1F TCCCAGGCAGCTCTGAACSEQ ID NO:40 Set P1 Set P1R ACCATCTTGACCTCGCCC SEQ ID NO:41 Set P2 SetP2F GTTCGCTTGACTGACGGC SEQ ID NO:42 Set P2 Set P2R ATGAGCCGCTGCCACAC SEQID NO:43 Set P3 Set P3F CACCGAAACCCAGAGACC SEQ ID NO:44 Set P3 Set P3RCCAAAGACACATACTCGACCC SEQ ID NO:45

[0079] These primers were used to amplify genomic DNA (10 ng) undertouch-down conditions. Touch-down annealing temperatures started at 65°C. and ended at 55° C. (-T of −0.5° C. per cycle) for 20 cycles,followed by 15 cycles at 55° C. Because of the high GC content of thetemplate, the PCR reactions contained 2.5 M betaine (W. Henke, et al.,Nucleic Acids Res. 25, 3957 (1997)). Reaction products were mixed 50:50with denaturing dyes (95% formamide, 10 mM NaOH, 20 mM EDTA, 0.02%bromophenol blue and 0.02% xylene cyanole) and heated to 95° C. for 5min irnmediately before loading. Samples were separated at 8 W for 16 hron 0.5×MDE (FMC) gels containing 0.6×Tris-borate buffer and 10%glycerol. Bands were visualized by silver staining.

[0080] A duplication of 12 nucleotides in the 5′ UTR was identified inRenal Cell Carcinoma (RCC) #1 (see FIG. 6A, lane 5) which was absent inmatched normal DNA and thus tumor-specific. This mutation was verifiedby multiple separate PCR amplifications, SSCP analyses and sequencing,as well as by the use of an alternative primer set, thus eliminating thepossibility of a PCR artifact. In the RCC #1 sample, very little of thewild-type heteroduplex product can be seen. This rearrangement resultedin an insertion of 12 bp in the tumor DNA (see FIG. 6B; bases 165 to 176of SEQ ID NO: 7), which was not present in the corresponding normal DNAof that patient. This insertion occurs in a consistently predictedstem-loop structure in the 5′ untranslated region (the RNA stem loopstructure was predicted by the GCG program MFOLD in both energeticallyoptimal and suboptimal folds). The consequence of this insertionconceivably affects either transcription or translation. Although thefrequency of TRC8 mutations in spontaneous tumors appears low, it ispossible this finding is reminiscent of -the mutation frequenciesobserved in BRCA1 and BRCA2 (P. A. Futreal, et al., Science 266, 120-122(1994); J. M. Lancaster, et al., Nat. Genet. 13, 238-240 (1996)).

[0081] Although this example is described in relation to one specificmutation, it should be appreciated that the general approach set forthcould be used to identify other mutations (including, for example, otherinsertions, or deletions, inversions and the like).

EXAMPLE 6 Amplification of TRC8 in a Sub-set of Variant Small Cell LungCarcinomas

[0082] Using Southern blot techniques, it was also shown that TRC8underwent a significant (6-fold) amplification in 1 of the 7 variantsmall-cell lung carcinoma cell lines which were tested (see FIG. 7).From available YAC contig data (http://www-genome.wi.mit.edu), it wasobserved that 3 intervening YACs are required to link TRC8 (880A9) andcMYC (934E1), thus the distance separating these genes must be on theorder of 2-3 Mb. To determine if the copy number increase resulted fromcoamplification of cMYC, the same blot was re-hybridized with the 380j9probe from this locus (26). While cMYC was amplified in two variant SCLClines (H82 and H524, lanes 10 and 16, respectively), it was onlyslightly increased in H211 (lane 12). These results indicate that TRC8may be amplified independently of MYC and suggest that TRC8 gain offunction may be important for tumorigenesis.

EXAMPLE 7 Synthesis of TRC8 Protein

[0083] The TRC8 protein was synthesized using the Promega in vitrotranscription/translation (TNT) kit. On ice, 25 ul of the TNT rabbitreticulocyte lysate were mixed with 2 ul of TNT reaction buffer, 1 ul ofRNA polymerase (T3 for sense; T7 for control antisense), 1 ul of theamino acid mixture, 2 ul of 35S-methionine (1000 Ci/mMole), 1 ul ofRNasin RNAase inhibitor (40 U/ul), 2 ul of the TRC8 cloned template(p45-1) and water to 50 ul total volume. Reactions were placed at 30° C.for 90 minutes. The reaction products were mixed 50:50 with laemmli SDSsample buffer and resolved on 7.0% SDS-polyacrylamide gels. It iscritical to NOT heat denature the TRC8 protein as this leads toirreversible aggregation and failure of gel resolution methods. The gelswere fixed in 10% acetic acid (30 min), neutralized with 0.1 M NaOH for10 min, impregnated with 1 M Na-salicylate and dried. Dried gels wereexposed to Kodak X-omat AR film without screens for 2 h to overnight.

EXAMPLE8 Diagnostic Applications

[0084] As noted above, evidence that various translocation events andmutations involving TRC8 appear to be associated with different tumorsand cancers make the detection of alterations to TRC8 a potentiallyuseful diagnostic tool. Diagnostic methods for identifying alterationscould involve several different approaches including, for example,direct mutation detection using TRC8 specific primers (for instance,those set forth as SEQ ID NO: 19-45, inclusively). The SSCP methodologydescribed in Example 5 is also a useful approach.

[0085] Since the present invention is described with specific referenceto the 3;8 translocation, methods for detecting their translocationevent are described below with regards to this particular translocation.It should be appreciated, however, that these approaches have moregeneral utility in detecting alterations to TRC8.

[0086] Thus, for example, the TRC8FHIT and reciprocal FHIT/TRC8 fusionsof the present invention can be used to determine the presence orabsence of chromosomal translocations within cells suspected of beingtumorous, especially within renal and thyroid cells Thus, the presentinvention can use PCR and DNA probes such as described above or YACs,cosmids and plasmids harboring the fused site to identify t(3;8) inrenal and thyroid cells. PCR has several advantages as a tool foridentifying t(3;8). PCR is rapid, sensitive and less affected by thequality of the samples as compared to chromosome methods such as FISHand karyotyping.

[0087] YACs and cosmids, however, can also be used as alternativediagnostic tools and have certain advantages as well. The YACs orcosmids can be quite specific since they preferably contain the fusedsite associated with the translocation. They may also yield a positiveresult in rare cases where PCR gives a negative result given that theYACs and cosmids contain a large region of the chromosome. Plasmidscontaining the DNA from the fused site can be used as probes to detectthe translocation by various hybridization methods, such as Southernblots for example.

[0088] PCR analysis of the translocation involves standard methods. RNAis isolated from cells according to standard protocols. cDNA is formedfrom the RNA template using reverse transcriptase. Using the cDNA andprimers specific for the t(3;8) such as described above, PCR is used toamplify the desired sequence. For example, a set of primers in which oneprimer binds at a point 5′ to the breakpoint and the second primer binds3′ to the breakpoint could be used to amplify the intervening sequence.The PCR products formed are separated by agarose gel electrophoresis andvisualized by well-known methods such as UV illumination after ethidiumbromide staining.

[0089] FISH can also be performed according to methods known to thoseskilled in the art. Typically, YACs or cosmid DNA can be labeled withbiotin. Metaphase chromosomes can be prepared from desired cells. Thebiotin-labeled probes are then allowed to hybridize with the chromosomesin the sample. The location of the hybridized areas can be detectedusing avidin with fluorescence tags and appropriate antibodies.

[0090] Southern blot hybridization can be performed by first isolatingDNA from a patient's cells. The DNA is then digested with restrictionendonucleases, the DNA fragments separated by gel electrophoresis andthe fragments then transferred to nylon membranes. Various radio-labeledprobes such as plasmids which contain the fused site can then be used tohybridize to DNA containing the breakpoint. Hybridization of the probeto DNA within the sample is typically done by autoradiography.

[0091] All the references listed herein, including, but not limited topatents and publications, are hereby incorporated by reference in theirentirety.

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 46 <210> SEQ ID NO 1<211> LENGTH: 2505 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220>FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (238)..(2232) <400>SEQUENCE: 1 gaaacccaga gacctcctgg ggagccgccg ccgccgccct ctcggccatcgctgcctccg 60 ccgcctgctc cacctcgagg gacgcgagcg ggcggcgggg ctggccgtgagagagacagg 120 agaggaagga gggcaggggc ggagttgccc gccttagccc ccgcccccggccgcggcccc 180 gggccctgcc ccgcgcggcc ctgcccggcc caccgagccc tggtgtggcagcggctc 237 atg gcg gcc gtg ggg ccc ccg cag cag cag gtg cgg atg gcc catcag 285 Met Ala Ala Val Gly Pro Pro Gln Gln Gln Val Arg Met Ala His Gln1 5 10 15 cag atc tgg gcg gcg ctc gaa gtg gcg ctc cgg gtg ccc tgc ctttac 333 Gln Ile Trp Ala Ala Leu Glu Val Ala Leu Arg Val Pro Cys Leu Tyr20 25 30 atc atc gac gcc atc ttc aac tcc tac ccg gat tcc agc caa agc cgg381 Ile Ile Asp Ala Ile Phe Asn Ser Tyr Pro Asp Ser Ser Gln Ser Arg 3540 45 ttc tgc atc gtg ctc cag atc ttc ctc cgg ctc ttt ggt gta ttt gca429 Phe Cys Ile Val Leu Gln Ile Phe Leu Arg Leu Phe Gly Val Phe Ala 5055 60 tcc agt att gtt ctg atc ttg tca caa cga tca ctt ttc aag ttt tac477 Ser Ser Ile Val Leu Ile Leu Ser Gln Arg Ser Leu Phe Lys Phe Tyr 6570 75 80 acg tac agc tca gcc ttt ctg tta gct gca act tca gtg ttg gtg aat525 Thr Tyr Ser Ser Ala Phe Leu Leu Ala Ala Thr Ser Val Leu Val Asn 8590 95 tat tat gct tct ttg cac att gac ttc tat ggt gcc tac aac acg tca573 Tyr Tyr Ala Ser Leu His Ile Asp Phe Tyr Gly Ala Tyr Asn Thr Ser 100105 110 gct ttt gga att gag ctg ctt cct cga aaa ggt ccc tcg ctg tgg atg621 Ala Phe Gly Ile Glu Leu Leu Pro Arg Lys Gly Pro Ser Leu Trp Met 115120 125 gca ctt atc gtt cta cag cta aca ttt gga att gga tac gtt aca cta669 Ala Leu Ile Val Leu Gln Leu Thr Phe Gly Ile Gly Tyr Val Thr Leu 130135 140 ctc cag att cat tcc atc tat tca caa tta att att ttg gat ctc ttg717 Leu Gln Ile His Ser Ile Tyr Ser Gln Leu Ile Ile Leu Asp Leu Leu 145150 155 160 gtt cct gta ata ggc tta atc aca gag cta cca tta cac atc agagag 765 Val Pro Val Ile Gly Leu Ile Thr Glu Leu Pro Leu His Ile Arg Glu165 170 175 act tta ctg ttt act tct tcc ttg att ctc aca tta aat aca gtgttt 813 Thr Leu Leu Phe Thr Ser Ser Leu Ile Leu Thr Leu Asn Thr Val Phe180 185 190 gtc ctg gca gtg aaa ctg aag tgg ttt tat tat tcc aca cga tatgtt 861 Val Leu Ala Val Lys Leu Lys Trp Phe Tyr Tyr Ser Thr Arg Tyr Val195 200 205 tat ctt ttg gtg agg cac atg tat cga att tat gga tta cag ttattg 909 Tyr Leu Leu Val Arg His Met Tyr Arg Ile Tyr Gly Leu Gln Leu Leu210 215 220 atg gag gac aca tgg aag agg att cgt ttc cca gac ata cta cgagtc 957 Met Glu Asp Thr Trp Lys Arg Ile Arg Phe Pro Asp Ile Leu Arg Val225 230 235 240 ttt tgg cta aca aga gtt aca gct cag gct aca gtg tta atgtac atc 1005 Phe Trp Leu Thr Arg Val Thr Ala Gln Ala Thr Val Leu Met TyrIle 245 250 255 tta agg atg gca aat gaa act gat tcc ttc ttt att tct tgggat gat 1053 Leu Arg Met Ala Asn Glu Thr Asp Ser Phe Phe Ile Ser Trp AspAsp 260 265 270 ttt tgg gac ctc att tgc aat ctt ata att agt ggg tgc gattct aca 1101 Phe Trp Asp Leu Ile Cys Asn Leu Ile Ile Ser Gly Cys Asp SerThr 275 280 285 cta act gta ctg ggc atg agt gct gta att tcc tca gta gcccat tat 1149 Leu Thr Val Leu Gly Met Ser Ala Val Ile Ser Ser Val Ala HisTyr 290 295 300 ttg ggg ctt gga ata ttg gcc ttt att gga tca act gag gaagat gac 1197 Leu Gly Leu Gly Ile Leu Ala Phe Ile Gly Ser Thr Glu Glu AspAsp 305 310 315 320 agg cgt ctt ggc ttt gtt gca cct gtt tta ttt ttt attttg gct ctt 1245 Arg Arg Leu Gly Phe Val Ala Pro Val Leu Phe Phe Ile LeuAla Leu 325 330 335 cag act ggg tta agt ggg cta aga cca gaa gag aga cttatt cgc tta 1293 Gln Thr Gly Leu Ser Gly Leu Arg Pro Glu Glu Arg Leu IleArg Leu 340 345 350 agt aga aac atg tgc ctt tta tta act gca gtc ctg catttt atc cat 1341 Ser Arg Asn Met Cys Leu Leu Leu Thr Ala Val Leu His PheIle His 355 360 365 gga atg aca gac cct gta tta atg tct ctc agt gcc tctcat gtg tca 1389 Gly Met Thr Asp Pro Val Leu Met Ser Leu Ser Ala Ser HisVal Ser 370 375 380 tct ttt cgt aga cat ttt cct gtg ctg ttt gtc tct gcttgc ctg ttt 1437 Ser Phe Arg Arg His Phe Pro Val Leu Phe Val Ser Ala CysLeu Phe 385 390 395 400 att ctt cct gtc tta ctc agt tat gtt ctt tgg catcac tat gca cta 1485 Ile Leu Pro Val Leu Leu Ser Tyr Val Leu Trp His HisTyr Ala Leu 405 410 415 aat aca tgg ttg ttt gca gtt aca gca ttt tgt gtggaa ctg tgc tta 1533 Asn Thr Trp Leu Phe Ala Val Thr Ala Phe Cys Val GluLeu Cys Leu 420 425 430 aaa gta att gtt tct ctc act gtt tat acg tta ttcatg att gat ggc 1581 Lys Val Ile Val Ser Leu Thr Val Tyr Thr Leu Phe MetIle Asp Gly 435 440 445 tac tat aat gtc ctc tgg gaa aag ctt gac gat tatgtc tac tac gtt 1629 Tyr Tyr Asn Val Leu Trp Glu Lys Leu Asp Asp Tyr ValTyr Tyr Val 450 455 460 cgt tca aca ggc agt att att gaa ttt ata ttt ggagtt gta atg ttt 1677 Arg Ser Thr Gly Ser Ile Ile Glu Phe Ile Phe Gly ValVal Met Phe 465 470 475 480 gga aat ggg gct tac act atg atg ttt gag tcggga agt aaa att cgg 1725 Gly Asn Gly Ala Tyr Thr Met Met Phe Glu Ser GlySer Lys Ile Arg 485 490 495 gct ttt atg atg tgc cta cat gca tat ttt aacatc tac tta caa gcc 1773 Ala Phe Met Met Cys Leu His Ala Tyr Phe Asn IleTyr Leu Gln Ala 500 505 510 aaa aat ggc tgg aag aca ttt atg aat cgt aggact gct gtg aag aaa 1821 Lys Asn Gly Trp Lys Thr Phe Met Asn Arg Arg ThrAla Val Lys Lys 515 520 525 att aat tca ctt cct gaa ata aaa ggg agc cgctta caa gaa ata aat 1869 Ile Asn Ser Leu Pro Glu Ile Lys Gly Ser Arg LeuGln Glu Ile Asn 530 535 540 gat gta tgt gca atc tgc tat cat gag ttt acaaca tct gct cgt att 1917 Asp Val Cys Ala Ile Cys Tyr His Glu Phe Thr ThrSer Ala Arg Ile 545 550 555 560 aca ccg tgt aat cat tat ttc cat gca ctttgc ctt cgg aaa tgg ctg 1965 Thr Pro Cys Asn His Tyr Phe His Ala Leu CysLeu Arg Lys Trp Leu 565 570 575 tac att caa gat act tgt cca atg tgc catcag aaa gta tac atc gaa 2013 Tyr Ile Gln Asp Thr Cys Pro Met Cys His GlnLys Val Tyr Ile Glu 580 585 590 gat gat atc aag gat aat tca aat gta tctaac aac aat gga ttt att 2061 Asp Asp Ile Lys Asp Asn Ser Asn Val Ser AsnAsn Asn Gly Phe Ile 595 600 605 cca ccc aat gaa act cca gag gaa gct gtaaga gaa gct gct gct gaa 2109 Pro Pro Asn Glu Thr Pro Glu Glu Ala Val ArgGlu Ala Ala Ala Glu 610 615 620 tct gac agg gaa ttg aac gaa gat gac agtaca gat tgt gat gat gat 2157 Ser Asp Arg Glu Leu Asn Glu Asp Asp Ser ThrAsp Cys Asp Asp Asp 625 630 635 640 gtt caa aga gaa aga aat gga gtg attcag cac aca ggc gca gca gct 2205 Val Gln Arg Glu Arg Asn Gly Val Ile GlnHis Thr Gly Ala Ala Ala 645 650 655 gaa gaa ttt aat gat gat act gac tgatgaaaatagc atttattaat 2252 Glu Glu Phe Asn Asp Asp Thr Asp 660 665gattgaggta tttgtttaaa attcagttca tccaaaatgg agtaatatcc ttcaccttca 2312gtgtgtaacc aagcacaaaa acagtatcaa tgttgaatct gtgaatggtt ttccgtttac 2372tgtgatgtgc tactgtaaat atacctcttt aattacttct ggtctctttg gtgacctgtt 2432taaatttgtg tacattattg tacatagaat aaaatgtttt cacattttta tgacaaaaaa 2492aaaaaaaaaa aaa 2505 <210> SEQ ID NO 2 <211> LENGTH: 664 <212> TYPE: PRT<213> ORGANISM: Homo sapiens <400> SEQUENCE: 2 Met Ala Ala Val Gly ProPro Gln Gln Gln Val Arg Met Ala His Gln 1 5 10 15 Gln Ile Trp Ala AlaLeu Glu Val Ala Leu Arg Val Pro Cys Leu Tyr 20 25 30 Ile Ile Asp Ala IlePhe Asn Ser Tyr Pro Asp Ser Ser Gln Ser Arg 35 40 45 Phe Cys Ile Val LeuGln Ile Phe Leu Arg Leu Phe Gly Val Phe Ala 50 55 60 Ser Ser Ile Val LeuIle Leu Ser Gln Arg Ser Leu Phe Lys Phe Tyr 65 70 75 80 Thr Tyr Ser SerAla Phe Leu Leu Ala Ala Thr Ser Val Leu Val Asn 85 90 95 Tyr Tyr Ala SerLeu His Ile Asp Phe Tyr Gly Ala Tyr Asn Thr Ser 100 105 110 Ala Phe GlyIle Glu Leu Leu Pro Arg Lys Gly Pro Ser Leu Trp Met 115 120 125 Ala LeuIle Val Leu Gln Leu Thr Phe Gly Ile Gly Tyr Val Thr Leu 130 135 140 LeuGln Ile His Ser Ile Tyr Ser Gln Leu Ile Ile Leu Asp Leu Leu 145 150 155160 Val Pro Val Ile Gly Leu Ile Thr Glu Leu Pro Leu His Ile Arg Glu 165170 175 Thr Leu Leu Phe Thr Ser Ser Leu Ile Leu Thr Leu Asn Thr Val Phe180 185 190 Val Leu Ala Val Lys Leu Lys Trp Phe Tyr Tyr Ser Thr Arg TyrVal 195 200 205 Tyr Leu Leu Val Arg His Met Tyr Arg Ile Tyr Gly Leu GlnLeu Leu 210 215 220 Met Glu Asp Thr Trp Lys Arg Ile Arg Phe Pro Asp IleLeu Arg Val 225 230 235 240 Phe Trp Leu Thr Arg Val Thr Ala Gln Ala ThrVal Leu Met Tyr Ile 245 250 255 Leu Arg Met Ala Asn Glu Thr Asp Ser PhePhe Ile Ser Trp Asp Asp 260 265 270 Phe Trp Asp Leu Ile Cys Asn Leu IleIle Ser Gly Cys Asp Ser Thr 275 280 285 Leu Thr Val Leu Gly Met Ser AlaVal Ile Ser Ser Val Ala His Tyr 290 295 300 Leu Gly Leu Gly Ile Leu AlaPhe Ile Gly Ser Thr Glu Glu Asp Asp 305 310 315 320 Arg Arg Leu Gly PheVal Ala Pro Val Leu Phe Phe Ile Leu Ala Leu 325 330 335 Gln Thr Gly LeuSer Gly Leu Arg Pro Glu Glu Arg Leu Ile Arg Leu 340 345 350 Ser Arg AsnMet Cys Leu Leu Leu Thr Ala Val Leu His Phe Ile His 355 360 365 Gly MetThr Asp Pro Val Leu Met Ser Leu Ser Ala Ser His Val Ser 370 375 380 SerPhe Arg Arg His Phe Pro Val Leu Phe Val Ser Ala Cys Leu Phe 385 390 395400 Ile Leu Pro Val Leu Leu Ser Tyr Val Leu Trp His His Tyr Ala Leu 405410 415 Asn Thr Trp Leu Phe Ala Val Thr Ala Phe Cys Val Glu Leu Cys Leu420 425 430 Lys Val Ile Val Ser Leu Thr Val Tyr Thr Leu Phe Met Ile AspGly 435 440 445 Tyr Tyr Asn Val Leu Trp Glu Lys Leu Asp Asp Tyr Val TyrTyr Val 450 455 460 Arg Ser Thr Gly Ser Ile Ile Glu Phe Ile Phe Gly ValVal Met Phe 465 470 475 480 Gly Asn Gly Ala Tyr Thr Met Met Phe Glu SerGly Ser Lys Ile Arg 485 490 495 Ala Phe Met Met Cys Leu His Ala Tyr PheAsn Ile Tyr Leu Gln Ala 500 505 510 Lys Asn Gly Trp Lys Thr Phe Met AsnArg Arg Thr Ala Val Lys Lys 515 520 525 Ile Asn Ser Leu Pro Glu Ile LysGly Ser Arg Leu Gln Glu Ile Asn 530 535 540 Asp Val Cys Ala Ile Cys TyrHis Glu Phe Thr Thr Ser Ala Arg Ile 545 550 555 560 Thr Pro Cys Asn HisTyr Phe His Ala Leu Cys Leu Arg Lys Trp Leu 565 570 575 Tyr Ile Gln AspThr Cys Pro Met Cys His Gln Lys Val Tyr Ile Glu 580 585 590 Asp Asp IleLys Asp Asn Ser Asn Val Ser Asn Asn Asn Gly Phe Ile 595 600 605 Pro ProAsn Glu Thr Pro Glu Glu Ala Val Arg Glu Ala Ala Ala Glu 610 615 620 SerAsp Arg Glu Leu Asn Glu Asp Asp Ser Thr Asp Cys Asp Asp Asp 625 630 635640 Val Gln Arg Glu Arg Asn Gly Val Ile Gln His Thr Gly Ala Ala Ala 645650 655 Glu Glu Phe Asn Asp Asp Thr Asp 660 <210> SEQ ID NO 3 <211>LENGTH: 1286 <212> TYPE: PRT <213> ORGANISM: Drosophila melanogaster<400> SEQUENCE: 3 Met Asp Arg Asp Ser Leu Pro Arg Val Pro Asp Thr HisGly Asp Val 1 5 10 15 Val Asp Glu Lys Leu Phe Ser Asp Leu Tyr Ile ArgThr Ser Trp Val 20 25 30 Asp Ala Gln Val Ala Leu Asp Gln Ile Asp Lys GlyLys Ala Arg Gly 35 40 45 Ser Arg Thr Ala Ile Tyr Leu Arg Ser Val Phe GlnSer His Leu Glu 50 55 60 Thr Leu Gly Ser Ser Val Gln Lys His Ala Gly LysVal Leu Phe Val 65 70 75 80 Ala Ile Leu Val Leu Ser Thr Phe Cys Val GlyLeu Lys Ser Ala Gln 85 90 95 Ile His Ser Lys Val His Gln Leu Trp Ile GlnGlu Gly Gly Arg Leu 100 105 110 Glu Ala Glu Leu Ala Tyr Thr Gln Lys ThrIle Gly Glu Asp Glu Ser 115 120 125 Ala Thr His Gln Leu Leu Ile Gln ThrThr His Asp Pro Asn Ala Ser 130 135 140 Val Leu His Pro Gln Ala Leu LeuAla His Leu Glu Val Leu Val Lys 145 150 155 160 Ala Thr Ala Val Lys ValHis Leu Tyr Asp Thr Glu Trp Gly Leu Arg 165 170 175 Asp Met Cys Asn MetPro Ser Thr Pro Ser Phe Glu Gly Ile Tyr Tyr 180 185 190 Ile Glu Gln IleLeu Arg His Leu Ile Pro Cys Ser Ile Ile Thr Pro 195 200 205 Leu Asp CysPhe Trp Glu Gly Ser Gln Leu Leu Gly Pro Glu Ser Ala 210 215 220 Val ValIle Pro Gly Leu Asn Gln Arg Leu Leu Trp Thr Thr Leu Asn 225 230 235 240Pro Ala Ser Val Met Gln Tyr Met Lys Gln Lys Met Ser Glu Glu Lys 245 250255 Ile Ser Phe Asp Phe Glu Thr Val Glu Gln Tyr Met Lys Arg Ala Ala 260265 270 Ile Gly Ser Gly Tyr Met Glu Lys Pro Cys Leu Asn Pro Leu Asn Pro275 280 285 Asn Cys Pro Asp Thr Ala Pro Asn Lys Asn Ser Thr Gln Pro ProAsp 290 295 300 Val Gly Ala Ile Leu Ser Gly Gly Cys Tyr Gly Tyr Ala AlaLys His 305 310 315 320 Met His Trp Pro Glu Glu Leu Ile Val Gly Gly ArgLys Arg Asn Arg 325 330 335 Ser Gly His Leu Arg Lys Ala Gln Ala Leu GlnSer Val Val Gln Leu 340 345 350 Met Thr Glu Lys Glu Met Tyr Asp Gln TrpGln Asp Asn Tyr Lys Val 355 360 365 His His Leu Gly Trp Thr Gln Glu LysAla Ala Glu Val Leu Asn Ala 370 375 380 Trp Gln Arg Asn Phe Ser Arg GluVal Glu Gln Leu Leu Arg Lys Gln 385 390 395 400 Ser Arg Ile Ala Thr AsnTyr Asp Ile Tyr Val Phe Ser Ser Ala Ala 405 410 415 Leu Asp Asp Ile LeuAla Lys Phe Ser His Pro Ser Ala Leu Ser Ile 420 425 430 Val Ile Gly ValAla Val Thr Val Leu Tyr Ala Phe Cys Thr Leu Leu 435 440 445 Arg Trp ArgAsp Pro Val Arg Gly Gln Ser Ser Val Gly Val Ala Gly 450 455 460 Val LeuLeu Met Cys Phe Ser Thr Ala Ala Gly Leu Gly Leu Ser Ala 465 470 475 480Leu Leu Gly Ile Val Phe Asn Ala Ala Ser Thr Gln Val Val Pro Phe 485 490495 Leu Ala Leu Gly Leu Gly Val Asp His Ile Phe Met Leu Thr Ala Ala 500505 510 Tyr Ala Glu Ser Asn Arg Arg Glu Gln Thr Lys Leu Ile Leu Lys Lys515 520 525 Val Gly Pro Ser Ile Leu Phe Ser Ala Cys Ser Thr Ala Gly SerPhe 530 535 540 Phe Ala Ala Ala Phe Ile Pro Val Pro Ala Leu Lys Val PheCys Leu 545 550 555 560 Gln Ala Ala Ile Val Met Cys Ser Asn Leu Ala AlaAla Leu Leu Val 565 570 575 Phe Pro Ala Met Ile Ser Leu Asp Leu Arg ArgArg Thr Ala Gly Arg 580 585 590 Ala Asp Ile Phe Cys Cys Cys Phe Pro ValTrp Lys Glu Gln Pro Lys 595 600 605 Val Ala Pro Pro Val Leu Pro Leu AsnAsn Asn Asn Gly Arg Gly Ala 610 615 620 Arg His Pro Lys Ser Cys Asn AsnAsn Arg Val Pro Leu Pro Ala Gln 625 630 635 640 Asn Pro Leu Leu Glu GlnArg Ala Asp Ile Pro Gly Ser Ser His Ser 645 650 655 Leu Ala Ser Phe SerLeu Ala Thr Phe Ala Phe Gln His Tyr Thr Pro 660 665 670 Phe Leu Met ArgSer Trp Val Lys Phe Leu Thr Val Met Gly Phe Leu 675 680 685 Ala Ala LeuIle Ser Ser Leu Tyr Ala Ser Thr Arg Leu Gln Asp Gly 690 695 700 Leu AspIle Ile Asp Leu Val Pro Lys Asp Ser Asn Glu His Lys Phe 705 710 715 720Leu Asp Ala Gln Thr Arg Leu Phe Gly Phe Tyr Ser Met Tyr Ala Val 725 730735 Thr Gln Gly Asn Phe Glu Tyr Pro Thr Gln Gln Gln Leu Leu Arg Asp 740745 750 Tyr His Asp Ser Phe Val Arg Val Pro His Val Ile Lys Asn Asp Asn755 760 765 Gly Gly Leu Pro Asp Phe Trp Leu Leu Leu Phe Ser Glu Trp LeuGly 770 775 780 Asn Leu Gln Lys Ile Phe Asp Glu Glu Tyr Arg Asp Gly ArgLeu Thr 785 790 795 800 Lys Glu Cys Trp Phe Pro Asn Ala Ser Ser Asp AlaIle Leu Ala Tyr 805 810 815 Lys Leu Ile Val Gln Thr Gly His Val Asp AsnPro Val Asp Lys Glu 820 825 830 Leu Val Leu Thr Asn Arg Leu Val Asn SerAsp Gly Ile Ile Asn Gln 835 840 845 Arg Ala Phe Tyr Asn Tyr Leu Ser AlaTrp Ala Thr Asn Asp Val Phe 850 855 860 Ala Tyr Gly Ala Ser Gln Gly LysLeu Tyr Pro Glu Pro Arg Gln Tyr 865 870 875 880 Phe His Gln Pro Asn GluTyr Asp Leu Lys Ile Pro Lys Ser Leu Pro 885 890 895 Leu Val Tyr Ala GlnMet Pro Phe Tyr Leu His Gly Leu Thr Asp Thr 900 905 910 Ser Gln Ile LysThr Leu Ile Gly His Ile Arg Asp Leu Ser Val Lys 915 920 925 Tyr Glu GlyPhe Gly Leu Pro Asn Tyr Pro Ser Gly Ile Pro Phe Ile 930 935 940 Phe TrpGlu Gln Tyr Met Thr Leu Arg Ser Ser Leu Ala Met Ile Leu 945 950 955 960Ala Cys Val Leu Leu Ala Ala Leu Val Leu Val Ser Leu Leu Leu Leu 965 970975 Ser Val Trp Ala Ala Val Leu Val Ile Leu Ser Val Leu Ala Ser Leu 980985 990 Ala Gln Ile Phe Gly Ala Met Thr Leu Leu Gly Ile Lys Leu Ser Ala995 1000 1005 Ile Pro Ala Val Ile Leu Ile Leu Ser Val Gly Met Met LeuCys Phe 1010 1015 1020 Asn Val Leu Ile Ser Leu Gly Phe Met Thr Ser ValGly Asn Arg Gln 1025 1030 1035 1040 Arg Arg Val Gln Leu Ser Met Gln MetSer Leu Gly Pro Leu Val His 1045 1050 1055 Gly Met Leu Thr Ser Gly ValAla Val Phe Met Leu Ser Thr Ser Pro 1060 1065 1070 Phe Glu Phe Val IleArg His Phe Cys Trp Leu Leu Leu Val Val Leu 1075 1080 1085 Cys Val GlyAla Cys Asn Ser Leu Leu Val Phe Pro Ile Leu Leu Ser 1090 1095 1100 MetVal Gly Pro Glu Ala Glu Leu Val Pro Leu Glu His Pro Asp Arg 1105 11101115 1120 Ile Ser Thr Pro Ser Pro Leu Pro Val Arg Ser Ser Lys Arg SerGly 1125 1130 1135 Lys Ser Tyr Val Val Gln Gly Ser Arg Ser Ser Arg GlySer Cys Gln 1140 1145 1150 Lys Ser His His His His His Lys Asp Leu AsnAsp Pro Ser Leu Thr 1155 1160 1165 Thr Ile Thr Glu Glu Pro Gln Ser TrpLys Ser Ser Asn Ser Ser Ile 1170 1175 1180 Gln Met Pro Asn Asp Trp ThrTyr Gln Pro Arg Glu Gln Arg Pro Ala 1185 1190 1195 1200 Ser Tyr Ala AlaPro Pro Pro Ala Tyr His Lys Ala Ala Ala Gln Gln 1205 1210 1215 His HisGln His Gln Gly Pro Pro Thr Thr Pro Pro Pro Pro Phe Pro 1220 1225 1230Thr Ala Tyr Pro Pro Glu Leu Gln Ser Ile Val Val Gln Pro Glu Val 12351240 1245 Thr Val Glu Thr Thr His Ser Asp Ser Asn Thr Thr Lys Val ThrAla 1250 1255 1260 Thr Ala Asn Ile Lys Val Glu Leu Ala Met Pro Gly ArgAla Val Arg 1265 1270 1275 1280 Ser Tyr Asn Phe Thr Ser 1285 <210> SEQID NO 4 <211> LENGTH: 888 <212> TYPE: PRT <213> ORGANISM: Homo sapiens<400> SEQUENCE: 4 Met Leu Ser Arg Leu Phe Arg Met His Gly Leu Phe ValAla Ser His 1 5 10 15 Pro Trp Glu Val Ile Val Gly Thr Val Thr Leu ThrIle Cys Met Met 20 25 30 Ser Met Asn Met Phe Thr Gly Asn Asn Lys Ile CysGly Trp Asn Tyr 35 40 45 Glu Cys Pro Lys Phe Glu Glu Asp Val Leu Ser SerAsp Ile Ile Ile 50 55 60 Leu Thr Ile Thr Arg Cys Ile Ala Ile Leu Tyr IleTyr Phe Gln Phe 65 70 75 80 Gln Asn Leu Arg Gln Leu Gly Ser Lys Tyr IleLeu Gly Ile Ala Gly 85 90 95 Leu Phe Thr Ile Phe Ser Ser Phe Val Phe SerThr Val Val Ile His 100 105 110 Phe Leu Asp Lys Glu Leu Thr Gly Leu AsnGlu Ala Leu Pro Phe Phe 115 120 125 Leu Leu Leu Ile Asp Leu Ser Arg AlaSer Thr Leu Ala Lys Phe Ala 130 135 140 Leu Ser Ser Asn Ser Gln Asp GluVal Arg Glu Asn Ile Ala Arg Gly 145 150 155 160 Met Ala Ile Leu Gly ProThr Phe Thr Leu Asp Ala Leu Val Glu Cys 165 170 175 Leu Val Ile Gly ValGly Thr Met Ser Gly Val Arg Gln Leu Glu Ile 180 185 190 Met Cys Cys PheGly Cys Met Ser Val Leu Ala Asn Tyr Phe Val Phe 195 200 205 Met Thr PhePhe Pro Ala Cys Val Ser Leu Val Leu Glu Leu Ser Arg 210 215 220 Glu SerArg Glu Gly Arg Pro Ile Trp Gln Leu Ser His Phe Ala Arg 225 230 235 240Val Leu Glu Glu Glu Glu Asn Lys Pro Asn Pro Val Thr Gln Arg Val 245 250255 Lys Met Ile Met Ser Leu Gly Leu Val Leu Val His Ala His Ser Arg 260265 270 Trp Ile Ala Asp Pro Ser Pro Gln Asn Ser Thr Ala Asp Thr Ser Lys275 280 285 Val Ser Leu Gly Leu Asp Glu Asn Val Ser Lys Arg Ile Glu ProSer 290 295 300 Val Ser Leu Trp Gln Phe Tyr Leu Ser Lys Met Ile Ser MetAsp Ile 305 310 315 320 Glu Gln Val Ile Thr Leu Ser Leu Ala Leu Leu LeuAla Val Lys Tyr 325 330 335 Ile Phe Phe Glu Gln Thr Glu Thr Glu Ser ThrLeu Ser Leu Lys Asn 340 345 350 Pro Ile Thr Ser Pro Val Val Thr Gln LysLys Val Pro Asp Asn Cys 355 360 365 Cys Arg Arg Glu Pro Met Leu Val ArgAsn Asn Gln Lys Cys Asp Ser 370 375 380 Val Glu Glu Glu Thr Gly Ile AsnArg Glu Arg Lys Val Glu Val Ile 385 390 395 400 Lys Pro Leu Val Ala GluThr Asp Thr Pro Asn Arg Ala Thr Phe Val 405 410 415 Val Gly Asn Ser SerLeu Leu Asp Thr Ser Ser Val Leu Val Thr Gln 420 425 430 Glu Pro Glu IleGlu Leu Pro Arg Glu Pro Arg Pro Asn Glu Glu Cys 435 440 445 Leu Gln IleLeu Gly Asn Ala Glu Lys Gly Ala Lys Phe Leu Ser Asp 450 455 460 Ala GluIle Ile Gln Leu Val Asn Ala Lys His Ile Pro Ala Tyr Lys 465 470 475 480Leu Glu Thr Leu Met Glu Thr His Glu Arg Gly Val Ser Ile Arg Arg 485 490495 Gln Leu Leu Ser Lys Lys Leu Ser Glu Pro Ser Ser Leu Gln Tyr Leu 500505 510 Pro Tyr Arg Asp Tyr Asn Tyr Ser Leu Val Met Gly Ala Cys Cys Glu515 520 525 Asn Val Ile Gly Tyr Met Pro Ile Pro Val Gly Val Ala Gly ProLeu 530 535 540 Cys Leu Asp Glu Lys Glu Phe Gln Val Pro Met Ala Thr ThrGlu Gly 545 550 555 560 Cys Leu Val Ala Ser Thr Asn Arg Gly Cys Arg AlaIle Gly Leu Gly 565 570 575 Gly Gly Ala Ser Ser Arg Val Leu Ala Asp GlyMet Thr Arg Gly Pro 580 585 590 Val Val Arg Leu Pro Arg Ala Cys Asp SerAla Glu Val Lys Ala Trp 595 600 605 Leu Glu Thr Ser Glu Gly Phe Ala ValIle Lys Glu Ala Phe Asp Ser 610 615 620 Thr Ser Arg Phe Ala Arg Leu GlnLys Leu His Thr Ser Ile Ala Gly 625 630 635 640 Arg Asn Leu Tyr Ile ArgPhe Gln Ser Arg Ser Gly Asp Ala Met Gly 645 650 655 Met Asn Met Ile SerLys Gly Thr Glu Lys Ala Leu Ser Lys Leu His 660 665 670 Glu Tyr Phe ProGlu Met Gln Ile Leu Ala Val Ser Gly Asn Tyr Cys 675 680 685 Thr Asp LysLys Pro Ala Ala Ile Asn Trp Ile Glu Gly Arg Gly Lys 690 695 700 Ser ValVal Cys Glu Ala Val Ile Pro Ala Lys Val Val Arg Glu Val 705 710 715 720Leu Lys Thr Thr Thr Glu Ala Met Ile Glu Val Asn Ile Asn Lys Asn 725 730735 Leu Val Gly Ser Ala Met Ala Gly Ser Ile Gly Gly Tyr Asn Ala His 740745 750 Ala Ala Asn Ile Val Thr Ala Ile Tyr Ile Ala Cys Gly Gln Asp Ala755 760 765 Ala Gln Asn Val Gly Ser Ser Asn Cys Ile Thr Leu Met Glu AlaSer 770 775 780 Gly Pro Thr Asn Glu Asp Leu Tyr Ile Ser Cys Thr Met ProSer Ile 785 790 795 800 Glu Ile Gly Thr Val Gly Gly Gly Thr Asn Leu LeuPro Gln Gln Ala 805 810 815 Cys Leu Gln Met Leu Gly Val Gln Gly Ala CysLys Asp Asn Pro Gly 820 825 830 Glu Asn Ala Arg Gln Leu Ala Arg Ile ValCys Gly Thr Val Met Ala 835 840 845 Gly Glu Leu Ser Leu Met Ala Ala LeuAla Ala Gly His Leu Val Lys 850 855 860 Ser His Met Ile His Asn Arg SerLys Ile Asn Leu Gln Asp Leu Gln 865 870 875 880 Gly Ala Cys Thr Lys LysThr Ala 885 <210> SEQ ID NO 5 <211> LENGTH: 1447 <212> TYPE: PRT <213>ORGANISM: Homo sapiens <400> SEQUENCE: 5 Met Ala Ser Ala Gly Asn Ala AlaGlu Pro Gln Asp Arg Gly Gly Gly 1 5 10 15 Gly Ser Gly Cys Ile Gly AlaPro Gly Arg Pro Ala Gly Gly Gly Arg 20 25 30 Arg Arg Arg Thr Gly Gly LeuArg Arg Ala Ala Ala Pro Asp Arg Asp 35 40 45 Tyr Leu His Arg Pro Ser TyrCys Asp Ala Ala Phe Ala Leu Glu Gln 50 55 60 Ile Ser Lys Gly Lys Ala ThrGly Arg Lys Ala Pro Leu Trp Leu Arg 65 70 75 80 Ala Lys Phe Gln Arg LeuLeu Phe Lys Leu Gly Cys Tyr Ile Gln Lys 85 90 95 Asn Cys Gly Lys Phe LeuVal Val Gly Leu Leu Ile Phe Gly Ala Phe 100 105 110 Ala Val Gly Leu LysAla Ala Asn Leu Glu Thr Asn Val Glu Glu Leu 115 120 125 Trp Val Glu ValGly Gly Arg Val Ser Arg Glu Leu Asn Tyr Thr Arg 130 135 140 Gln Lys IleGly Glu Glu Ala Met Phe Asn Pro Gln Leu Met Ile Gln 145 150 155 160 ThrPro Lys Glu Glu Gly Ala Asn Val Leu Thr Thr Glu Ala Leu Leu 165 170 175Gln His Leu Asp Ser Ala Leu Gln Ala Ser Arg Val His Val Tyr Met 180 185190 Tyr Asn Arg Gln Trp Lys Leu Glu His Leu Cys Tyr Lys Ser Gly Glu 195200 205 Leu Ile Thr Glu Thr Gly Tyr Met Asp Gln Ile Ile Glu Tyr Leu Tyr210 215 220 Pro Cys Leu Ile Ile Thr Pro Leu Asp Cys Phe Trp Glu Gly AlaLys 225 230 235 240 Leu Gln Ser Gly Thr Ala Tyr Leu Leu Gly Lys Pro ProLeu Arg Trp 245 250 255 Thr Asn Phe Asp Pro Leu Glu Phe Leu Glu Glu LeuLys Lys Ile Asn 260 265 270 Tyr Gln Val Asp Ser Trp Glu Glu Met Leu AsnLys Ala Glu Val Gly 275 280 285 His Gly Tyr Met Asp Arg Pro Cys Leu AsnPro Ala Asp Pro Asp Cys 290 295 300 Pro Ala Thr Ala Pro Asn Lys Asn SerThr Lys Pro Leu Asp Met Ala 305 310 315 320 Leu Val Leu Asn Gly Gly CysHis Gly Leu Ser Arg Lys Tyr Met His 325 330 335 Trp Gln Glu Glu Leu IleVal Gly Gly Thr Val Lys Asn Ser Thr Gly 340 345 350 Lys Leu Val Ser AlaHis Ala Leu Gln Thr Met Phe Gln Leu Met Thr 355 360 365 Pro Lys Gln MetTyr Glu His Phe Lys Gly Tyr Glu Tyr Val Ser His 370 375 380 Ile Asn TrpAsn Glu Asp Lys Ala Ala Ala Ile Leu Glu Ala Trp Gln 385 390 395 400 ArgThr Tyr Val Glu Val Val His Gln Ser Val Ala Gln Asn Ser Thr 405 410 415Gln Lys Val Leu Ser Phe Thr Thr Thr Thr Leu Asp Asp Ile Leu Lys 420 425430 Ser Phe Ser Asp Val Ser Val Ile Arg Val Ala Ser Gly Tyr Leu Leu 435440 445 Met Leu Ala Tyr Ala Cys Leu Thr Met Leu Arg Trp Asp Cys Ser Lys450 455 460 Ser Gln Gly Ala Val Gly Leu Ala Gly Val Leu Leu Val Ala LeuSer 465 470 475 480 Val Ala Ala Gly Leu Gly Leu Cys Ser Leu Ile Gly IleSer Phe Asn 485 490 495 Ala Ala Thr Thr Gln Val Leu Pro Phe Leu Ala LeuGly Val Gly Val 500 505 510 Asp Asp Val Phe Leu Leu Ala His Ala Phe SerGlu Thr Gly Gln Asn 515 520 525 Lys Arg Ile Pro Phe Glu Asp Arg Thr GlyGlu Cys Leu Lys Arg Thr 530 535 540 Gly Ala Ser Val Ala Leu Thr Ser IleSer Asn Val Thr Ala Phe Phe 545 550 555 560 Met Ala Ala Leu Ile Pro IlePro Ala Leu Arg Ala Phe Ser Leu Gln 565 570 575 Ala Ala Val Val Val ValPhe Asn Phe Ala Met Val Leu Leu Ile Phe 580 585 590 Pro Ala Ile Leu SerMet Asp Leu Tyr Arg Arg Glu Asp Arg Arg Leu 595 600 605 Asp Ile Phe CysCys Phe Thr Ser Pro Cys Val Ser Arg Val Ile Gln 610 615 620 Val Glu ProGln Ala Tyr Thr Asp Thr His Asp Asn Thr Arg Tyr Ser 625 630 635 640 ProPro Pro Pro Tyr Ser Ser His Ser Phe Ala His Glu Thr Gln Ile 645 650 655Thr Met Gln Ser Thr Val Gln Leu Arg Thr Glu Tyr Asp Pro His Thr 660 665670 His Val Tyr Tyr Thr Thr Ala Glu Pro Arg Ser Glu Ile Ser Val Gln 675680 685 Pro Val Thr Val Thr Gln Asp Thr Leu Ser Cys Gln Ser Pro Glu Ser690 695 700 Thr Ser Ser Thr Arg Asp Leu Leu Ser Gln Phe Ser Asp Ser SerLeu 705 710 715 720 His Cys Leu Glu Pro Pro Cys Thr Lys Trp Thr Leu SerSer Phe Ala 725 730 735 Glu Lys His Tyr Ala Pro Phe Leu Leu Lys Pro LysAla Lys Val Val 740 745 750 Val Ile Phe Leu Phe Leu Gly Leu Leu Gly ValSer Leu Tyr Gly Thr 755 760 765 Thr Arg Val Arg Asp Gly Leu Asp Leu ThrAsp Ile Val Pro Arg Glu 770 775 780 Thr Arg Glu Tyr Asp Phe Ile Ala AlaGln Phe Lys Tyr Phe Ser Phe 785 790 795 800 Tyr Asn Met Tyr Ile Val ThrGln Lys Ala Asp Tyr Pro Asn Ile Gln 805 810 815 His Leu Leu Tyr Asp LeuHis Arg Ser Phe Ser Asn Val Lys Tyr Val 820 825 830 Met Leu Glu Glu AsnLys Gln Leu Pro Lys Met Trp Leu His Tyr Phe 835 840 845 Arg Asp Trp LeuGln Gly Leu Gln Asp Ala Phe Asp Ser Asp Trp Glu 850 855 860 Thr Gly LysIle Met Pro Asn Asn Tyr Lys Asn Gly Ser Asp Asp Gly 865 870 875 880 ValLeu Ala Tyr Lys Leu Leu Val Gln Thr Gly Ser Arg Asp Lys Pro 885 890 895Ile Asp Ile Ser Gln Leu Thr Lys Gln Arg Leu Val Asp Ala Asp Gly 900 905910 Ile Ile Asn Pro Ser Ala Phe Tyr Ile Tyr Leu Thr Ala Trp Val Ser 915920 925 Asn Asp Pro Val Ala Tyr Ala Ala Ser Gln Ala Asn Ile Arg Pro His930 935 940 Arg Pro Glu Trp Val His Asp Lys Ala Asp Tyr Met Pro Glu ThrArg 945 950 955 960 Leu Arg Ile Pro Ala Ala Glu Pro Ile Glu Tyr Ala GlnPhe Pro Phe 965 970 975 Tyr Leu Asn Gly Leu Arg Asp Thr Ser Asp Phe ValGlu Ala Ile Glu 980 985 990 Lys Val Arg Thr Ile Cys Ser Asn Tyr Thr SerLeu Gly Leu Ser Ser 995 1000 1005 Tyr Pro Asn Gly Tyr Pro Phe Leu PheTrp Glu Gln Tyr Ile Gly Leu 1010 1015 1020 Arg His Trp Leu Leu Leu PheIle Ser Val Val Leu Ala Cys Thr Phe 1025 1030 1035 1040 Leu Val Cys AlaVal Phe Leu Leu Asn Pro Trp Thr Ala Gly Ile Ile 1045 1050 1055 Val MetVal Leu Ala Leu Met Thr Val Glu Leu Phe Gly Met Met Gly 1060 1065 1070Leu Ile Gly Ile Lys Leu Ser Ala Val Pro Val Val Ile Leu Ile Ala 10751080 1085 Ser Val Gly Ile Gly Val Glu Phe Thr Val His Val Ala Leu AlaPhe 1090 1095 1100 Leu Thr Ala Ile Gly Asp Lys Asn Arg Arg Ala Val LeuAla Leu Glu 1105 1110 1115 1120 His Met Phe Ala Pro Val Leu Asp Gly AlaVal Ser Thr Leu Leu Gly 1125 1130 1135 Val Leu Met Leu Ala Gly Ser GluPhe Asp Phe Ile Val Arg Tyr Phe 1140 1145 1150 Phe Ala Val Leu Ala IleLeu Thr Ile Leu Gly Val Leu Asn Gly Leu 1155 1160 1165 Val Leu Leu ProVal Leu Leu Ser Phe Phe Gly Pro Tyr Pro Glu Val 1170 1175 1180 Ser ProAla Asn Gly Leu Asn Arg Leu Pro Thr Pro Ser Pro Glu Pro 1185 1190 11951200 Pro Pro Ser Val Val Arg Phe Ala Met Pro Pro Gly His Thr His Ser1205 1210 1215 Gly Ser Asp Ser Ser Asp Ser Glu Tyr Ser Ser Gln Thr ThrVal Ser 1220 1225 1230 Gly Leu Ser Glu Glu Leu Arg His Tyr Glu Ala GlnGln Gly Ala Gly 1235 1240 1245 Gly Pro Ala His Gln Val Ile Val Glu AlaThr Glu Asn Pro Val Phe 1250 1255 1260 Ala His Ser Thr Val Val His ProGlu Ser Arg His His Pro Pro Ser 1265 1270 1275 1280 Asn Pro Arg Gln GlnPro His Leu Asp Ser Gly Ser Leu Pro Pro Gly 1285 1290 1295 Arg Gln GlyGln Gln Pro Arg Arg Asp Pro Pro Arg Glu Gly Leu Trp 1300 1305 1310 ProPro Leu Tyr Arg Pro Arg Arg Asp Ala Phe Glu Ile Ser Thr Glu 1315 13201325 Gly His Ser Gly Pro Ser Asn Arg Ala Arg Trp Gly Pro Arg Gly Ala1330 1335 1340 Arg Ser His Asn Pro Arg Asn Pro Ala Ser Thr Ala Met GlySer Ser 1345 1350 1355 1360 Val Pro Gly Tyr Cys Gln Pro Ile Thr Thr ValThr Ala Ser Ala Ser 1365 1370 1375 Val Thr Val Ala Val His Pro Pro ProVal Pro Gly Pro Gly Arg Asn 1380 1385 1390 Pro Arg Gly Gly Leu Cys ProGly Tyr Pro Glu Thr Asp His Gly Leu 1395 1400 1405 Phe Glu Asp Pro HisVal Pro Phe His Val Arg Cys Glu Arg Arg Asp 1410 1415 1420 Ser Lys ValGlu Val Ile Glu Leu Gln Asp Val Glu Cys Glu Glu Arg 1425 1430 1435 1440Pro Arg Gly Ser Ser Ser Asn 1445 <210> SEQ ID NO 6 <211> LENGTH: 5605<212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221>NAME/KEY: unsure <222> LOCATION: (5021) <221> NAME/KEY: unsure <222>LOCATION: (5097) <221> NAME/KEY: unsure <222> LOCATION: (5166) <221>NAME/KEY: unsure <222> LOCATION: (5297) <221> NAME/KEY: unsure <222>LOCATION: (5391) <400> SEQUENCE: 6 actcgatctt tttccccata atggcctctctccttttgct ttacagtgca atcagggcag 60 aataacagat atgttgagta gctaatttatatttctgaag atctccatag aactcttaag 120 ttctaagtct tcatagtctt tgcaccttgccaagtttgct ttcctttggg aattctgtca 180 taaccacaag ggttatcccc agacatgcagtttggggaaa ttaacccggc agtagtaaac 240 agactggatt agaaaaggtg gcagtcgtaataaggatatt caataaccgg ggaaagctac 300 agaaggatct tgagttagga tgatgacagttacaatagaa aggagaccag tacaagagac 360 ggtgaaaaca gaatggatac aatttagttgaagaattggc tgtgggtgaa aagaagaaat 420 caaagatgcc tgaagttgtg agtttcaggtagccctgagt ttataatcct agttctgact 480 cgtactatct ttggatatac tagctgctgactactaactg aacaaggtag taaccttgtc 540 atcttgtaac ttacctaagt ctgtttcctcctctataaaa caaactatca aaataaagcc 600 gatgaaaaga ttgaagtaaa acataaaacccttagcacag tgcctagtat ccattaagca 660 gtagataaat gatggcggtc attattagggagtagggtaa agaacagaag gttaaaaaca 720 tgagtagcag taaggacttt tgggtggggattcggtcttt ctactacaca ttacacatac 780 ctctaagcag agaaaggggt tggaggggagtttgataatg tgactaataa tcctcctccc 840 cctggggggg atcttgataa aggatagccacccacatgga ggcctaatat aaactggatt 900 gcagaatgca cttgagctgg gagcccaagcgggccaggct tcttgattcc tattttatcc 960 cctttaacct aggatagcta ggtctatagtgcagactcag acctaactta cactttcatt 1020 tgcagctgaa gtttcggaac aaagacaaagatagccagat catattaatt acacggatag 1080 gcaagaaagc atgagccctg aggaggaaggaagggactgt ccaggtgtac ttacctcaaa 1140 gatgagaaat atcaaagaca ggaaaccctaggttcttgcc cttcagtcgc tatctccttg 1200 ccattagtaa aatgcggccg atgaatgtcctcacttctgt ccatctgggc aggaggtggg 1260 aagggtgacg tgcaaatgga tgggaggaacccttttttcg gcagcaccca ccacacccag 1320 cctagtgcca cgcaccgcaa gcgctccataaacgcacaca gcgtcgcctc taccaggatc 1380 ccgggcggcc ttcgcgggat ttctcctggcgtcggctttc agactcccga gggtgggata 1440 aatcgagagg gtggcatcct ttggcttttcttctcccagg cagctctgaa ccatgtttat 1500 gcaacgttta atgggctcta ataaaacggctaataatttt gatccgcgga agcaccgact 1560 cgctcgctaa gccgaatctg cgagggtgaagctgcaactc caacgccgga aagcgcggct 1620 accgaaaagc gcatgcgcca cggggtggcacgaagctaga gtaagctgag gaggtgggcg 1680 gaaaccatgg caaccatggg tgatgacgacatggggagcg tctctacgct ggattatgac 1740 gctggattat gacgcaggca gtgggcgcggactctgcggt tcgcttgact gacggcgcag 1800 cctccgggcc tagccacagc agcaacggcagaggccagcg ggcgaggtca agatggtggc 1860 tccgcgggcg ggggaggcag tggagggaggaggagtcaga ccttagccag ccggaaacac 1920 cgaaacccag agacctcctg gggagccgccgccgccgccc tctcggccat cgctgcctcc 1980 gccgcctgct ccacctcgag ggacgcgagcgggcggcggg gctggccgtg agagagacag 2040 gagaggaagg agggcagggg cggagttgcccgccttagcc cccgcccccg gccgcggccc 2100 cgggccctgc cccgcgcggc cctgcccggcccaccgagcc ctggtgtggc agcggctcat 2160 ggcggccgtg gggcccccgc agcagcaggtgcggatggcc catcagcaga tctgggcggc 2220 gctcgaagtg gcgctccggg tgccctgcctttacatcatc gacgccatct tcaactccta 2280 cccggattcc agccaaagcc ggttctgcatcgtgctccag atcttcctcc ggctctttgg 2340 taagggaaca gggtaccgta cgtcccgggacggctatgcg ggccgagacg ttccccgggg 2400 agcgggcagg cgcgcagagg ccatgggtcgagtatgtgtc tttgggaact acagtttcat 2460 ccctcaaagg gtcgtcttta cgggttgggagggggaagga gatgatactt gtctgagttg 2520 acagcggagc agtcccccta gcgaggattcagtgttgtac ctgagtgagg tggcgagaaa 2580 gtaaggatgt gtgagcatgc agctctttgcccgtttgcct tgggaaatga gtactggctt 2640 accgcgtcct atgcgacagc cttcgaaaaacaactctgct atcccagtct cgtcgtgtgt 2700 gaccttggac ttctgccaag gcgtgggcctcagtttccct ttattaccaa atgccgagta 2760 cttgtatgtt gtcacttctg ctgcacccgagggtccacag gatcgtgaag tgtctgcagt 2820 aaatgacatc caagcgaagg agacagggttttgaatgtca gaccttgaaa ccattaccaa 2880 aaattctcat gaaagcggcg atgcttaaatcgagtcctaa aattttccat gaataaacga 2940 gaaggtattg aaagtgactg taaaaagtgatgagacattt gccaaagaga cttctgaaaa 3000 tcattgtctt tgtttcgtat ggatggagtgttaaaagtat cttatatttc atttcttcct 3060 ttccatcttc acttacatca tcagagggagatgcatttat gcgtaaaata attttttaag 3120 taattgtaac ttggcaaatt ttgttgatgaggtaattctg gtgatcggtc ttaagtaggc 3180 tagtgctttt gaagtgttgc tgagggcagactgatggtga ggtctttacc aagcctttct 3240 gaattacaag ccatctgttg ctttattccttcaaaagagg agtggcaata aaggccgaat 3300 tttgttctta gcaataaata gccatattttacatttatct ggtattttcc ttttgcaaag 3360 cattttcact ggtaggtatt atctaacctaaccttcaaac atcccccaga ggtgggtggt 3420 ggtattgtca ttttacaaac caggaaacagggtcatagag gttaagtgtt ttgctcttgg 3480 taggctgccg ctaagtggtt gccagagcctgagcttaaaa atcttttgac attaaatatg 3540 gtttactttc ctccctaaca gctgcaatccagatacagaa tgcagtagta atgatcatat 3600 gctagatctt aaaactaaag atgtaaacgtgatgttcagc ccaagctgct tcagtaaccc 3660 acctgctacc taattccagc ctttcctacttgccactgct aacgtatacc accaccataa 3720 tttgtgttaa aaactgggct ctgtttgagaggattctgca tgttcatccc agaaacagtg 3780 tagtgactgc tttgaatatt cttacagcatgataactgca gattctgtga gaatcccttt 3840 gggacctggt gctttaatgc agtagtctgttgagcttcag gttaggcagc ttcaaggaaa 3900 agaagtattt tctttaataa ataatttgtagtgtaagaac attgaacaaa gcaatctgat 3960 attttccttg tactaggcat gtttcttttgtaataaatct tggaagtttg tgtttgacaa 4020 agaacaacat gacttaaggc aactgctcagtgctttggtt tcttattttt accttttaaa 4080 agatggagag tgaggttatc tgtttcatagggtgttaaag acaataaatc tattttaatg 4140 cttcatttaa taccatattt tgttagatattttggtcttt tgaaatcatc ggcaaaatat 4200 tggtgttgtt tatttgaaaa ttttgatccctggaatctct ggatcagact ttgagaaata 4260 cgaaatactc ttcaaatcta tcatccgactcttcttactg tgtcatgtaa ttatgctaat 4320 ttttcacatt tgtaaggatg ataagaatgaaaaaaactga tttcggtgct aaaaattatg 4380 ttactaaaag acacaggttt gtgtgtaaatatgccaatgg atagttgaaa ttaatcgttc 4440 tgccttgtta gtttttttgt agttgtagacttctcagtac tactgtgtat aagtcttacc 4500 acttgtcaac ctcacttttt tctttaaaattaatgtgtaa aaatggaaat ctctaagtaa 4560 aaagcattac cagttttatg tgtggtggtagaagtattta ctcccctcta cctacaaaat 4620 ggtgctacaa ctgtaccttc atttgatatcaaactaattt gagatttcaa ccggttagaa 4680 aataaagcaa ctcattttgg agatccaaataaaattctat gacaatatat ggaatattga 4740 agaacataaa gctgtgtttt aagggatgttaaatactaaa cgaaactcaa cccagagcag 4800 cataatgctg agttagcaaa tttaaatccaaggcagaaaa tgcaagccta ggagtttgtt 4860 ttaatagtat tactaatatg gtgataattcattgaggtgg gaaaaggctt taaacataac 4920 agaagaccca gaagttccaa ggaaaaatctgaaaaatttc agtacataaa aataaaaagc 4980 ttttttatgg caaagcttaa aaaagaatccttaaatgaca nactgagata aaatacttct 5040 atcataagtg attcacgttc cagtaagagttttaccctgc cagggaagct gaggcangag 5100 aatcgcttga accaaggagt tgcaggttgcagtgagccga gatcgctcca ctgcactcca 5160 gcctgnggat agagtgagac tctgtctaaaaaaaaaaaca aaacaaaaca agaaaaaact 5220 tatactccct taggaaaatg cctaagacaagcaattcaca ataaacaaaa tgcagatgtt 5280 caataagcaa gagaaanagt atttagcccttcctgcagta gcttgatttt atgggggaaa 5340 aaaaaaagta ctagccttgt ttataattaaatactgtcaa gaccgcttac nggtgctcac 5400 gcctataatc ccagcacttt gggaggctgaggcaagcaga tttgcttatg ccaggagttg 5460 acaacagctt tggcaaatgg cacaacccgtgtcaacaaat aattaaccgg gtgcaatgac 5520 tcctggccat attccaacct acgggtttggagctttcaca taatttcttt attcagggag 5580 gttttagtgc cattagcatt atagt 5605<210> SEQ ID NO 7 <211> LENGTH: 2517 <212> TYPE: DNA <213> ORGANISM:Homo sapiens <220> FEATURE: <221> NAME/KEY: repeat_unit <222> LOCATION:(165)..(176) <400> SEQUENCE: 7 gaaacccaga gacctcctgg ggagccgccgccgccgccct ctcggccatc gctgcctccg 60 ccgcctgctc cacctcgagg gacgcgagcgggcggcgggg ctggccgtga gagagacagg 120 agaggaagga gggcaggggc ggagttgcccgccttagccc ccgccttagc ccccgccccc 180 ggccgcggcc ccgggccctg ccccgcgcggccctgcccgg cccaccgagc cctggtgtgg 240 cagcggctca tggcggccgt ggggcccccgcagcagcagg tgcggatggc ccatcagcag 300 atctgggcgg cgctcgaagt ggcgctccgggtgccctgcc tttacatcat cgacgccatc 360 ttcaactcct acccggattc cagccaaagccggttctgca tcgtgctcca gatcttcctc 420 cggctctttg gtgtatttgc atccagtattgttctgatct tgtcacaacg atcacttttc 480 aagttttaca cgtacagctc agcctttctgttagctgcaa cttcagtgtt ggtgaattat 540 tatgcttctt tgcacattga cttctatggtgcctacaaca cgtcagcttt tggaattgag 600 ctgcttcctc gaaaaggtcc ctcgctgtggatggcactta tcgttctaca gctaacattt 660 ggaattggat acgttacact actccagattcattccatct attcacaatt aattattttg 720 gatctcttgg ttcctgtaat aggcttaatcacagagctac cattacacat cagagagact 780 ttactgttta cttcttcctt gattctcacattaaatacag tgtttgtcct ggcagtgaaa 840 ctgaagtggt tttattattc cacacgatatgtttatcttt tggtgaggca catgtatcga 900 atttatggat tacagttatt gatggaggacacatggaaga ggattcgttt cccagacata 960 ctacgagtct tttggctaac aagagttacagctcaggcta cagtgttaat gtacatctta 1020 aggatggcaa atgaaactga ttccttctttatttcttggg atgatttttg ggacctcatt 1080 tgcaatctta taattagtgg gtgcgattctacactaactg tactgggcat gagtgctgta 1140 atttcctcag tagcccatta tttggggcttggaatattgg cctttattgg atcaactgag 1200 gaagatgaca ggcgtcttgg ctttgttgcacctgttttat tttttatttt ggctcttcag 1260 actgggttaa gtgggctaag accagaagagagacttattc gcttaagtag aaacatgtgc 1320 cttttattaa ctgcagtcct gcattttatccatggaatga cagaccctgt attaatgtct 1380 ctcagtgcct ctcatgtgtc atcttttcgtagacattttc ctgtgctgtt tgtctctgct 1440 tgcctgttta ttcttcctgt cttactcagttatgttcttt ggcatcacta tgcactaaat 1500 acatggttgt ttgcagttac agcattttgtgtggaactgt gcttaaaagt aattgtttct 1560 ctcactgttt atacgttatt catgattgatggctactata atgtcctctg ggaaaagctt 1620 gacgattatg tctactacgt tcgttcaacaggcagtatta ttgaatttat atttggagtt 1680 gtaatgtttg gaaatggggc ttacactatgatgtttgagt cgggaagtaa aattcgggct 1740 tttatgatgt gcctacatgc atattttaacatctacttac aagccaaaaa tggctggaag 1800 acatttatga atcgtaggac tgctgtgaagaaaattaatt cacttcctga aataaaaggg 1860 agccgcttac aagaaataaa tgatgtatgtgcaatctgct atcatgagtt tacaacatct 1920 gctcgtatta caccgtgtaa tcattatttccatgcacttt gccttcggaa atggctgtac 1980 attcaagata cttgtccaat gtgccatcagaaagtataca tcgaagatga tatcaaggat 2040 aattcaaatg tatctaacaa caatggatttattccaccca atgaaactcc agaggaagct 2100 gtaagagaag ctgctgctga atctgacagggaattgaacg aagatgacag tacagattgt 2160 gatgatgatg ttcaaagaga aagaaatggagtgattcagc acacaggcgc agcagctgaa 2220 gaatttaatg atgatactga ctgatgaaaatagcatttat taatgattga ggtatttgtt 2280 taaaattcag ttcatccaaa atggagtaatatccttcacc ttcagtgtgt aaccaagcac 2340 aaaaacagta tcaatgttga atctgtgaatggttttccgt ttactgtgat gtgctactgt 2400 aaatatacct ctttaattac ttctggtctctttggtgacc tgtttaaatt tgtgtacatt 2460 attgtacata gaataaaatg ttttcacatttttatgacaa aaaaaaaaaa aaaaaaa 2517 <210> SEQ ID NO 8 <211> LENGTH: 1095<212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221>NAME/KEY: CDS <222> LOCATION: (363)..(806) <400> SEQUENCE: 8 tccccgctctgctctgtccg gtcacaggac tttttgccct ctgttcccgg gtccctcagg 60 cggccacccagtgggcacac tcccaggcgg cgctccggcc ccgcgctccc tccctctgcc 120 tttcattcccagctgtcaac atcctggaag ctttgaagct caggaaagaa gagaaatcca 180 ctgagaacagtctgtaaagg tccgtagtgc tatctacatc cagacggtgg aagggagaga 240 aagagaaagaaggtatccta ggaatacctg cctgcttaga ccctctataa aagctctgtg 300 catcctgccactgaggactc cgaagaggta gcagtcttct gaaagacttc aactgtgagg 360 ac atg tcgttc aga ttt ggc caa cat ctc atc aag ccc tct gta gtg 407 Met Ser Phe ArgPhe Gly Gln His Leu Ile Lys Pro Ser Val Val 1 5 10 15 ttt ctc aaa acagaa ctg tcc ttc gct ctt gtg aat agg aaa cct gtg 455 Phe Leu Lys Thr GluLeu Ser Phe Ala Leu Val Asn Arg Lys Pro Val 20 25 30 gta cca gga cat gtcctt gtg tgc ccg ctg cgg cca gtg gag cgc ttc 503 Val Pro Gly His Val LeuVal Cys Pro Leu Arg Pro Val Glu Arg Phe 35 40 45 cat gac ctg cgt cct gatgaa gtg gcc gat ttg ttt cag acg acc cag 551 His Asp Leu Arg Pro Asp GluVal Ala Asp Leu Phe Gln Thr Thr Gln 50 55 60 aga gtc ggg aca gtg gtg gaaaaa cat ttc cat ggg acc tct ctc acc 599 Arg Val Gly Thr Val Val Glu LysHis Phe His Gly Thr Ser Leu Thr 65 70 75 ttt tcc atg cag gat ggc ccc gaagcc gga cag act gtg aag cac gtt 647 Phe Ser Met Gln Asp Gly Pro Glu AlaGly Gln Thr Val Lys His Val 80 85 90 95 cac gtc cat gtt ctt ccc agg aaggct gga gac ttt cac agg aat gac 695 His Val His Val Leu Pro Arg Lys AlaGly Asp Phe His Arg Asn Asp 100 105 110 agc atc tat gag gag ctc cag aaacat gac aag gag gac ttt cct gcc 743 Ser Ile Tyr Glu Glu Leu Gln Lys HisAsp Lys Glu Asp Phe Pro Ala 115 120 125 tct tgg aga tca gag gag gaa atggca gca gaa gcc gca gct ctg cgg 791 Ser Trp Arg Ser Glu Glu Glu Met AlaAla Glu Ala Ala Ala Leu Arg 130 135 140 gtc tac ttt cag tga cacagatgtttttcagatcc tgaattccag caaaagagct 846 Val Tyr Phe Gln 145 attgccaaccagtttgaaga ccgccccccc gcctctcccc aagaggaact gaatcagcat 906 gaaaatgcagtttcttcatc tcaccatcct gtattcttca accagtgatc ccccacctcg 966 gtcactccaactcccttaaa atacctagac ctaaacggct cagacaggca gatttgaggt 1026 ttccccctgtctccttattc ggcagcctta tgattaaact tccttctctg ctgcaaaaaa 1086 aaaaaaaaa1095 <210> SEQ ID NO 9 <211> LENGTH: 147 <212> TYPE: PRT <213> ORGANISM:Homo sapiens <400> SEQUENCE: 9 Met Ser Phe Arg Phe Gly Gln His Leu IleLys Pro Ser Val Val Phe 1 5 10 15 Leu Lys Thr Glu Leu Ser Phe Ala LeuVal Asn Arg Lys Pro Val Val 20 25 30 Pro Gly His Val Leu Val Cys Pro LeuArg Pro Val Glu Arg Phe His 35 40 45 Asp Leu Arg Pro Asp Glu Val Ala AspLeu Phe Gln Thr Thr Gln Arg 50 55 60 Val Gly Thr Val Val Glu Lys His PheHis Gly Thr Ser Leu Thr Phe 65 70 75 80 Ser Met Gln Asp Gly Pro Glu AlaGly Gln Thr Val Lys His Val His 85 90 95 Val His Val Leu Pro Arg Lys AlaGly Asp Phe His Arg Asn Asp Ser 100 105 110 Ile Tyr Glu Glu Leu Gln LysHis Asp Lys Glu Asp Phe Pro Ala Ser 115 120 125 Trp Arg Ser Glu Glu GluMet Ala Ala Glu Ala Ala Ala Leu Arg Val 130 135 140 Tyr Phe Gln 145<210> SEQ ID NO 10 <211> LENGTH: 1261 <212> TYPE: DNA <213> ORGANISM:Homo sapiens <400> SEQUENCE: 10 gaaacccaga gacctcctgg ggagccgccgccgccgccct ctcggccatc gctgcctccg 60 ccgcctgctc cacctcgagg gacgcgagcgggcggcgggg ctggccgtga gagagacagg 120 agaggaagga gggcaggggc ggagttgcccgccttagccc ccgcccccgg ccgcggcccc 180 gggccctgcc ccgcgcggcc ctgcccggcccaccgagccc tggtgtggca gcggctcatg 240 gcggccgtgg ggcccccgca gcagcaggtgcggatggccc atcagcagat ctgggcggcg 300 ctcgaagtgg cgctccgggt gccctgcctttacatcatcg acgccatctt caactcctac 360 ccggattcca gccaaagccg gttctgcatcgtgctccaga tcttcctccg gctctttggt 420 atcctaggaa tacctgcctg cttagaccctctataaaagc tctgtgcatc ctgccactga 480 ggactccgaa gaggtagcag tcttctgaaagacttcaact gtgaggacat gtcgttcaga 540 tttggccaac atctcatcaa gccctctgtagtgtttctca aaacagaact gtccttcgct 600 cttgtgaata ggaaacctgt ggtaccaggacatgtccttg tgtgcccgct gcggccagtg 660 gagcgcttcc atgacctgcg tcctgatgaagtggccgatt tgtttcagac gacccagaga 720 gtcgggacag tggtggaaaa acatttccatgggacctctc tcaccttttc catgcaggat 780 ggccccgaag ccggacagac tgtgaagcacgttcacgtcc atgttcttcc caggaaggct 840 ggagactttc acaggaatga cagcatctatgaggagctcc agaaacatga caaggaggac 900 tttcctgcct cttggagatc agaggaggaaatggcagcag aagccgcagc tctgcgggtc 960 tactttcagt gacacagatg tttttcagatcctgaattcc agcaaaagag ctattgccaa 1020 ccagtttgaa gaccgccccc ccgcctctccccaagaggaa ctgaatcagc atgaaaatgc 1080 agtttcttca tctcaccatc ctgtattcttcaaccagtga tcccccacct cggtcactcc 1140 aactccctta aaatacctag acctaaacggctcagacagg cagatttgag gtttccccct 1200 gtctccttat tcggcagcct tatgattaaacttccttctc tgctgcaaaa aaaaaaaaaa 1260 a 1261 <210> SEQ ID NO 11 <211>LENGTH: 2339 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400>SEQUENCE: 11 tccccgctct gctctgtccg gtcacaggac tttttgccct ctgttcccgggtccctcagg 60 cggccaccca gtgggcacac tcccaggcgg cgctccggcc ccgcgctccctccctctgcc 120 tttcattccc agctgtcaac atcctggaag ctttgaagct caggaaagaagagaaatcca 180 ctgagaacag tctgtaaagg tccgtagtgc tatctacatc cagacggtggaagggagaga 240 aagagaaaga aggtgtattt gcatccagta ttgttctgat cttgtcacaacgatcacttt 300 tcaagtttta cacgtacagc tcagcctttc tgttagctgc aacttcagtgttggtgaatt 360 attatgcttc tttgcacatt gacttctatg gtgcctacaa cacgtcagcttttggaattg 420 agctgcttcc tcgaaaaggt ccctcgctgt ggatggcact tatcgttctacagctaacat 480 ttggaattgg atacgttaca ctactccaga ttcattccat ctattcacaattaattattt 540 tggatctctt ggttcctgta ataggcttaa tcacagagct accattacacatcagagaga 600 ctttactgtt tacttcttcc ttgattctca cattaaatac agtgtttgtcctggcagtga 660 aactgaagtg gttttattat tccacacgat atgtttatct tttggtgaggcacatgtatc 720 gaatttatgg attacagtta ttgatggagg acacatggaa gaggattcgtttcccagaca 780 tactacgagt cttttggcta acaagagtta cagctcaggc tacagtgttaatgtacatct 840 taaggatggc aaatgaaact gattccttct ttatttcttg ggatgatttttgggacctca 900 tttgcaatct tataattagt gggtgcgatt ctacactaac tgtactgggcatgagtgctg 960 taatttcctc agtagcccat tatttggggc ttggaatatt ggcctttattggatcaactg 1020 aggaagatga caggcgtctt ggctttgttg cacctgtttt attttttattttggctcttc 1080 agactgggtt aagtgggcta agaccagaag agagacttat tcgcttaagtagaaacatgt 1140 gccttttatt aactgcagtc ctgcatttta tccatggaat gacagaccctgtattaatgt 1200 ctctcagtgc ctctcatgtg tcatcttttc gtagacattt tcctgtgctgtttgtctctg 1260 cttgcctgtt tattcttcct gtcttactca gttatgttct ttggcatcactatgcactaa 1320 atacatggtt gtttgcagtt acagcatttt gtgtggaact gtgcttaaaagtaattgttt 1380 ctctcactgt ttatacgtta ttcatgattg atggctacta taatgtcctctgggaaaagc 1440 ttgacgatta tgtctactac gttcgttcaa caggcagtat tattgaatttatatttggag 1500 ttgtaatgtt tggaaatggg gcttacacta tgatgtttga gtcgggaagtaaaattcggg 1560 cttttatgat gtgcctacat gcatatttta acatctactt acaagccaaaaatggctgga 1620 agacatttat gaatcgtagg actgctgtga agaaaattaa ttcacttcctgaaataaaag 1680 ggagccgctt acaagaaata aatgatgtat gtgcaatctg ctatcatgagtttacaacat 1740 ctgctcgtat tacaccgtgt aatcattatt tccatgcact ttgccttcggaaatggctgt 1800 acattcaaga tacttgtcca atgtgccatc agaaagtata catcgaagatgatatcaagg 1860 ataattcaaa tgtatctaac aacaatggat ttattccacc caatgaaactccagaggaag 1920 ctgtaagaga agctgctgct gaatctgaca gggaattgaa cgaagatgacagtacagatt 1980 gtgatgatga tgttcaaaga gaaagaaatg gagtgattca gcacacaggcgcagcagctg 2040 aagaatttaa tgatgatact gactgatgaa aatagcattt attaatgattgaggtatttg 2100 tttaaaattc agttcatcca aaatggagta atatccttca ccttcagtgtgtaaccaagc 2160 acaaaaacag tatcaatgtt gaatctgtga atggttttcc gtttactgtgatgtgctact 2220 gtaaatatac ctctttaatt acttctggtc tctttggtga cctgtttaaatttgtgtaca 2280 ttattgtaca tagaataaaa tgttttcaca tttttatgac aaaaaaaaaaaaaaaaaaa 2339 <210> SEQ ID NO 12 <211> LENGTH: 22 <212> TYPE: DNA <213>ORGANISM: Homo sapiens <400> SEQUENCE: 12 tcagaagact gctacctctt cg 22<210> SEQ ID NO 13 <211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: Homosapiens <400> SEQUENCE: 13 tcagtggcag gatgcacag 19 <210> SEQ ID NO 14<211> LENGTH: <212> TYPE: <213> ORGANISM: <400> SEQUENCE: 14 000 <210>SEQ ID NO 15 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Homosapiens <400> SEQUENCE: 15 ggtctaagca ggcaggtatt c 21 <210> SEQ ID NO 16<211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400>SEQUENCE: 16 tggaagggag agaaagag 18 <210> SEQ ID NO 17 <211> LENGTH: 16<212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 17ggtattccta ggatac 16 <210> SEQ ID NO 18 <211> LENGTH: 19 <212> TYPE: DNA<213> ORGANISM: Homo sapiens <400> SEQUENCE: 18 tccctctgcc tttcattcc 19<210> SEQ ID NO 19 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Homosapiens <400> SEQUENCE: 19 gccctgcctt tacatcatcg ac 22 <210> SEQ ID NO20 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400>SEQUENCE: 20 agatctggag cacgatgcag aac 23 <210> SEQ ID NO 21 <211>LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE:21 tcttgttagc caaaagactc g 21 <210> SEQ ID NO 22 <211> LENGTH: 17 <212>TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 22 agttgcccgccttagcc 17 <210> SEQ ID NO 23 <211> LENGTH: 21 <212> TYPE: DNA <213>ORGANISM: Homo sapiens <400> SEQUENCE: 23 ccaaagacac atactcgacc c 21<210> SEQ ID NO 24 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Homosapiens <400> SEQUENCE: 24 cataactctt agtggggaaa cattc 25 <210> SEQ IDNO 25 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Homo sapiens<400> SEQUENCE: 25 tgtaacgtat ccaattccaa atg 23 <210> SEQ ID NO 26 <211>LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE:26 tggcacttat cgttctacag c 21 <210> SEQ ID NO 27 <211> LENGTH: 21 <212>TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 27 tcttgttagccaaaagactc g 21 <210> SEQ ID NO 28 <211> LENGTH: 19 <212> TYPE: DNA<213> ORGANISM: Homo sapiens <400> SEQUENCE: 28 agtgtttgtc ctggcagtg 19<210> SEQ ID NO 29 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Homosapiens <400> SEQUENCE: 29 acagttagtg tagaatcgca ccc 23 <210> SEQ ID NO30 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400>SEQUENCE: 30 tggcaaatga aactgattcc 20 <210> SEQ ID NO 31 <211> LENGTH:21 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 31catggataaa atgcaggact g 21 <210> SEQ ID NO 32 <211> LENGTH: 24 <212>TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 32 aagaccagaagagagactta ttcg 24 <210> SEQ ID NO 33 <211> LENGTH: 20 <212> TYPE: DNA<213> ORGANISM: Homo sapiens <400> SEQUENCE: 33 tgctgtaact gcaaacaacc 20<210> SEQ ID NO 34 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Homosapiens <400> SEQUENCE: 34 tctttggcat cactatgcac 20 <210> SEQ ID NO 35<211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400>SEQUENCE: 35 cttcacagca gtcctacgat tc 22 <210> SEQ ID NO 36 <211>LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE:36 ccaaaaatgg ctggaagac 19 <210> SEQ ID NO 37 <211> LENGTH: 19 <212>TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 37 tgtcagattcagcagcagc 19 <210> SEQ ID NO 38 <211> LENGTH: 19 <212> TYPE: DNA <213>ORGANISM: Homo sapiens <400> SEQUENCE: 38 ccacccaatg aaactccag 19 <210>SEQ ID NO 39 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Homosapiens <400> SEQUENCE: 39 agtagcacat cacagtaaac gg 22 <210> SEQ ID NO40 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400>SEQUENCE: 40 tcccaggcag ctctgaac 18 <210> SEQ ID NO 41 <211> LENGTH: 18<212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 41accatcttga cctcgccc 18 <210> SEQ ID NO 42 <211> LENGTH: 18 <212> TYPE:DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 42 gttcgcttga ctgacggc18 <210> SEQ ID NO 43 <211> LENGTH: 17 <212> TYPE: DNA <213> ORGANISM:Homo sapiens <400> SEQUENCE: 43 atgagccgct gccacac 17 <210> SEQ ID NO 44<211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400>SEQUENCE: 44 caccgaaacc cagagacc 18 <210> SEQ ID NO 45 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 45ccaaagacac atactcgacc c 21 <210> SEQ ID NO 46 <211> LENGTH: 61 <212>TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence:Ring-finger motif of theRING-H2 subtype <221> NAME/KEY: UNSURE <222> LOCATION: (2)..(3) <223>OTHER INFORMATION: Must be exactly two Xaa at this location; Xaa can beany amino acid <221> NAME/KEY: UNSURE <222> LOCATION: (5)..(31) <223>OTHER INFORMATION: Must be at least 9 Xaa residues in this region and asmany as 27 xaa residues, where Xaa is any amino acid. <221> NAME/KEY:UNSURE <222> LOCATION: (33) <221> NAME/KEY: UNSURE <222> LOCATION:(35)..(36) <223> OTHER INFORMATION: Must be exactly 2 Xaa at thislocation; Xaa can be any amino acid. <221> NAME/KEY: UNSURE <222>LOCATION: (38)..(39) <223> OTHER INFORMATION: Must be exactly 2 Xaa atthis location; Xaa can be any amino acid. <221> NAME/KEY: UNSURE <222>LOCATION: (41)..(57) <223> OTHER INFORMATION: At least 6 Xaa must bepresent in this region up to a maximum of 17 Xaa. Xaa can be any aminoacid. <221> NAME/KEY: UNSURE <222> LOCATION: (59)..(60) <223> OTHERINFORMATION: Must be exactly 2 Xaa at this positon; Xaa can be any aminoacid. <400> SEQUENCE: 46 Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Cys 20 25 30 Xaa His Xaa Xaa His Xaa Xaa Cys Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys XaaXaa Cys 50 55 60

We claim:
 1. An isolated nucleic acid molecule encoding the polypeptidecomprising the amino acid sequence of SEQ ID NO:
 2. 2. The nucleic acidmolecule of claim 1, wherein said nucleic acid molecule comprises thenucleotide sequence of SEQ ID NO:
 1. 3. The nucleic acid molecule ofclaim 1, wherein said nucleic acid-molecule comprises nucleotides 238 to2229 of SEQ ID NO:
 1. 4. An isolated nucleic acid molecule comprising anucleotide sequence selected from the group consisting of (a) adeoxyribonucleotide sequence complementary to nucleotides 238 to 2229 ofSEQ ID NO: 1; (b) a ribonucleotide sequence complementary to nucleotides238 to 2229 of SEQ ID NO: 1; (c) a nucleotide sequence complementary tothe deoxyribonucleotide sequence of (a) or to the ribonucleotidesequence of (b); (d) a nucleotide sequence of at least 12 consecutivenucleotides capable of hybridizing to nucleotides 238 to 2229 of SEQ IDNO: 1; and (e) a nucleotide sequence capable of hybridizing to anucleotide sequence of (d).
 5. A vector containing the nucleic acidmolecule of claim
 1. 6. A host cell containing the vector of claim
 5. 7.A method for detecting the presence of TRC8 gene in a biological sample,comprising: (a) contacting a nucleic acid probe which is at least 12continuous nucleotides in length and is specific for binding to humanTRC8 gene with said sample under conditions which allow said nucleicacid probe to anneal to complementary sequences in said sample; and (b)detecting duplex formation between said nucleic acid probe and saidcomplementary sequences.
 8. A method according to claim 7, wherein saidnucleic acid probe used in said contacting step is a subsequence of theentire human TRC8 gene.
 9. An isolated DNA molecule comprising anucleotide sequence selected from the group of SEQ ID NO: 19 to SEQ IDNO: 45, inclusively.
 10. A method for producing a polypeptide comprisingthe amino acid sequence of SEQ ID NO: 2 or fragments thereof, saidmethod comprising the steps of: (a) culturing said host cell of claim 6under conditions suitable for the expression of the polypeptide; and (b)recovering the polypeptide from the host cell culture.
 11. A polypeptideproduct of the expression in a host cell of a DNA according to themethod of claim
 10. 12. An isolated nucleic acid molecule of SEQ ID NO:6.
 13. An isolated nucleic acid molecule comprising a nucleotidesequence selected from the group consisting of (a) a deoxyribonucleotidesequence complementary to SEQ ID NO: 6; (b) a ribonucleotide sequencecomplementary to SEQ ID NO: 6; (c) a nucleotide sequence complementaryto the deoxyribonucleotide sequence of (a) or to the ribonucleotidesequence of (b); (d) a nucleotide sequence of at least 12 consecutivenucleotides capable of hybridizing to nucleotides of SEQ ID NO: 6; and(e) a nucleotide sequence capable of hybridizing to a nucleotidesequence of (d).
 14. An isolated nucleic acid molecule of SEQ ID NO: 7.15. The nucleic acid molecule of claim 14, wherein said nucleic acidmolecule comprises nucleotides 153 to 176 of SEQ ID NO:
 7. 16. Anisolated nucleic acid molecule comprising a nucleotide sequence selectedfrom the group consisting of (a) a deoxyribonucleotide sequencecomplementary to nucleotides 153 to 176 of SEQ ID NO: 7; (b) aribonucleotide sequence complementary to nucleotides 153 to 176 of SEQID NO: 7; and (c) a nucleotide sequence complementary to thedeoxyribonucleotide sequence of (a) or to the ribonucleotide sequence of(b).
 17. An isolated nucleic acid molecule comprising a nucleotidesequence selected from the group consisting of (a) a deoxyribonucleotidesequence of SEQ ID NO: 10; (b) a deoxyribonucleotide sequencecomplementary to the deoxyribonucleotide sequence of (a); (c) aribonucleotide sequence complementary to the deoxyribonucleotidesequence of (a); and (d) a nucleotide sequence complementary to thedeoxyribonucleotide sequence of (b) or to the ribonucleotide sequence of(c).
 18. A nucleic acid probe selected from the group consisting of (a)a deoxyribonucleotide sequence which is a DNA fragment comprisingcontiguous nucleotides on the 5′ and 3′ sides of the fused site ofTRC8FHIT fused DNA which has the nucleotide sequence of SEQ ID NO: 10(b) a ribonucleotide sequence complementary to said deoxyribonucleotidesequence of (a); and (c) a nucleotide sequence complementary to thedeoxyribonucleotide sequence of (a) or to the ribonucleotide sequence of(b), wherein the fused site is between bases 418 and 419 of thenucleotide sequence of SEQ ID NO:
 10. 19. A nucleic acid probe accordingto claim 18, wherein said probe is a DNA fragment comprising contiguousnucleotides on the 5′ and 3′ sides of the fused site of TRC8/FHIT fusedDNA, the fused site being the site between bases 418 and 419 of thenucleotide sequence of SEQ ID NO: 10, wherein said DNA fragmentspecifically hybridizes with the nucleotide sequence of SEQ ID NO: 10but does not specifically hybridize with TRC8 DNA or FHIT DNA.
 20. Apair of oligonucleotides wherein one of the oligonucleotidesspecifically hybridizes with the TRC8/FHIT fused DNA comprising thecontiguous nucleotide sequence of SEQ ID NO: 10 on the 3′ side of afused site and the other oligonucleotide specifically hybridizes withthe TRC8/FHIT fused DNA on the 5′ side of said fused site, said fusedsite being located between bases 418 and 419 of SEQ ID NO:
 10. 21. Anisolated nucleic acid molecule comprising a nucleotide sequence selectedfrom the group consisting of (a) a deoxyribonucleotide sequence of SEQID NO: 11; (b) a deoxyribonucleotide sequence complementary to thedeoxyribonucleotide sequence of (a); (c) a ribonucleotide sequencecomplementary to the deoxyribonucleotide sequence of (a); and (d) anucleotide sequence complementary to the deoxyribonucleotide sequence of(b) or to the ribonucleotide sequence of (c).
 22. A nucleic acid probeselected from the group consisting of (a) a deoxyribonucleotide sequencewhich is a DNA fragment comprising contiguous nucleotides on the 5′ and3′ sides of the fused site of FHIT/TRC8 fused DNA which has thenucleotide sequence of SEQ ID NO: 11; (b) a ribonucleotide sequencecomplementary to said deoxyribonucleotide sequence of (a); and (c) anucleotide sequence complementary to the deoxyribonucleotide sequence of(a) or to the ribonucleotide sequence of (b), wherein the fused site isbetween bases 252 and 253 of the nucleotide sequence of SEQ ID NO: 11.23. A nucleic acid probe according to claim 22, wherein said probe is aDNA fragment comprising contiguous nucleotides on the 5′ and 3′ sides ofthe fused site of FHIT/TRC8 fused DNA, said fused site being the sitebetween bases 252 and 253 of the nucleotide sequence of SEQ ID NO: 11,wherein said DNA fragment specifically hybridizes with the nucleotidesequence of SEQ ID NO: 11, but does not specifically hybridize with TRC8DNA or FHIT DNA.
 24. A pair of oligonucleotides wherein one of theoligonucleotides specifically hybridizes with the FHIT/TRC8 fused DNAcomprising the contiguous nucleotide sequence of SEQ ID NO: 11 on the 3′side of a fused site and the other oligonucleotide specificallyhybridizes with the FHIT/TRC8 fused DNA on the 5′ side of said fusedsite, said fused site being located between bases 252 and 253 of SEQ IDNO:
 11. 25. A method of diagnosing or assessing renal or thyroid tumorformation in humans, comprising determining whether a TRC8 gene has beenrearranged or mutated, a rearranged or mutated TRC8 gene indicating arenal or thyroid tumor.
 26. A method according to claim 25, wherein saiddetermining step involves ascertaining whether there is a breakpoint inthe TRC8 gene between bases 418 and 419 of the nucleotide sequence ofSEQ ID NO:
 1. 27. A method for detecting alterations to human TRC8,comprising: (a) amplifying the DNA in a human DNA sample to formamplification products; and (b) screening said amplification products todetect an alteration of TRC8.
 28. A method according to claim 27,wherein said amplifying step involves utilization of a primer having asequence selected from the group of SEQ ID NO: 19-45, inclusively.
 29. Amethod according to claim 27, wherein said screening step includesperforming single-stranded conformational polymorphism analysis.
 30. Amethod for detecting a 3;8 human chromosomal translocation, comprising:(a) contacting a nucleic acid probe of claim 18 with a biological sampleto be tested; (b) determining whether said nucleic acid probespecifically hybridizes to a nucleic acid molecule of claim
 17. 31. Amethod for detecting a 3;8 human chromosomal translocation, comprising:(a) contacting a nucleic acid probe of claim 22 with a biological sampleto be tested; (b) determining whether said nucleic acid probespecifically hybridizes to a nucleic acid molecule of claim
 21. 32. Amethod for detecting fused DNA containing the fused site of TRC8/FHITfused DNA, said fused site being the site between bases 418 and 419 ofthe nucleotide sequence of SEQ ID NO: 10, comprising the steps of: (a)contacting said probe of claim 18 with a sample to be tested; and (b)determining whether said probe specifically hybridizes with said fusedDNA in said sample but not with TRC8 DNA or FHIT DNA.
 33. A method fordetecting fused DNA containing the fused site of FHIT/TRC8 fused DNA,said fused site being the site between bases 252 and 253 of thenucleotide sequence of SEQ ID NO: 11, comprising the steps of: (a)contacting said probe of claim 22 with a sample to be tested; and (b)determining whether said probe specifically hybridizes with said fusedDNA in said sample but not with TRC8 DNA or FHIT DNA.